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 Advances in
CANCER
RESEARCH

Volume 86
This Page Intentionally Left Blank
 Advances in
CANCER
RESEARCH
Volume 86

Edited by

George F. Vande Woude

Van Andel Research Institute
Grand Rapids, Michigan

George Klein
Microbiology and Tumor Biology Center
Karolinska Institute
Stockholm, Sweden

Amsterdam Boston London New York Oxford Paris

San Diego San Francisco Singapore Sydney Tokyo
This book is printed on acid-free paper. 
∞

Copyright 
C 2002, Elsevier Science (USA).

All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any
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02 03 04 05 06 07 MM 9 8 7 6 5 4 3 2 1
 Contents

Contributors to Volume 86 vii

Coordinate Regulation of Translation by the PI 3-Kinase

and mTOR Pathways
Kathleen A. Martin and John Blenis
I. Overview of Translational Regulatory Pathways 2
II. PI 3-K and mTOR Effectors which Regulate Translation 7
III. Regulation of Translational Effectors 10
IV. Coordinated Translational Control 20
V. Conclusions 29
References 30

Histone Acetyltransferases and Deacetylases in the Control

of Cell Proliferation and Differentiation
Heike Lehrmann, Linda Louise Pritchard, and Annick Harel-Bellan
I. Introduction 42
II. Acetylation of Histones 43
III. Histone Acetyltransferases 44
IV. Histone Deacetylases and Cell Cycle Regulation 48
V. Muscle Differentiation 51
VI. Hematopoiesis 53
VII. Huntington’s Disease 55
VIII. Histone Acetylation in Combination with Other Chromatin Modifications 56
IX. Conclusion 57
References 58

Molecular Pathogenesis of Human Hepatocellular Carcinoma

Michael A. Kern, Kai Breuhahn, and Peter Schirmacher
I. Introduction 67
II. Morphology of Human Hepatocarcinogenesis 69

v
vi Contents

III. Molecular Etiology 72

IV. Host Carcinogenic Events 77
V. Functional Consequences 88
VI. Therapeutic Implications 90
References 92

The Cell-Mediated Immune Response to Human

Papillomavirus-Induced Cervical Cancer: Implications
for Immunotherapy
Gretchen L. Eiben, Markwin P. Velders, and W. Martin Kast
I. Introduction 113
II. Human Papillomaviruses 114
III. Cellular Immunity to HPV 116
IV. Immunotherapy against HPV-Induced Carcinomas 123
V. Conclusion 136
References 137

The T-Cell Response in Patients with Cancer

Chiara Castelli and Markus J. Maeurer
I. Definition of Immune Effector Functions 149
II. Defining the Bait for Antigen-Specific T Cells 152
III. The Role of the Coreceptor CD8 in Mediating Antitumor
Restricted T-Cell Responses 158
IV. Tools to Measure ex Vivo T-Cell Avidity 160
V. Biomarkers or True Surrogate Markers? 163
VI. T-Cell Crossreactivity 172
VII. Questions of Specificity and Alternate T-Cell Effector Functions 173
References 176

The Life and Death of a B Cell

Thierry Defrance, Montserrat Casamayor-Pallejà, and Peter H. Krammer
I. Introduction 196
II. The Maintenance of B Cell Tolerance 197
III. The Regulation of B Cell Homeostasis 201
IV. Control of the Specificity and Affinity of the Ab Response 206
V. Regulation of Survival in the Memory B Cell Compartment 212
VI. Conclusive Remarks 216
References 218
 Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

John Blenis, Department of Cell Biology, Harvard Medical School, Boston,

Massachusetts 02115 (1)
Kai Breuhahn, Institute of Pathology, University of Cologne, D-50931
Cologne, Germany (67)
Montserrat Casamayor-Pallejà, INSERM U404, “Immunity and Vaccina-
tion,” 69365, Lyon, Cedex 07, France (195)
Chiara Castelli, Unit of Immunotherapy of Human Tumors, Istituto
Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy (149)
Thierry Defrance, INSERM U404, “Immunity and Vaccination,” 69365,
Lyon, Cedex 07, France (195)
Gretchen L. Eiben, Cardinal Bernardin Cancer Center, Loyola University
Chicago, Maywood, Illinois 60153 (113)
Annick Harel-Bellan, CNRS UPR 9079, Institut André Lwoff, 94800
Villejuif, France (41)
W. Martin Kast, Cardinal Bernardin Cancer Center, Loyola University
Chicago, Maywood, Illinois 60153 (113)
Michael A. Kern, Institute of Pathology, University of Cologne, D-50931
Cologne, Germany (67)
Peter H. Krammer, Tumor Immunology Program, German Cancer
Research Center, D-69120 Heidelberg, Germany (195)
Heike Lehrmann, CNRS UPR 9079, Institut André Lwoff, 94800 Villejuif,
France (41)
Markus J. Maeurer, Department of Medical Microbiology, University of
Mainz, 55101 Mainz, Germany (149)
Kathleen A. Martin, Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115 (1)∗
Linda Louise Pritchard, CNRS UPR 9079, Institut André Lwoff, 94800
Villejuif, France (41)

∗ Present address: Departments of Surgery and of Pharmacology and Toxicology, Dartmouth

Medical School, Lebanon, New Hampshire 03756

vii
viii Contributors

Peter Schirmacher, Institute of Pathology, University of Cologne, D-50931

Cologne, Germany (67)
Markwin P. Velders, Cardinal Bernardin Cancer Center, Loyola University
Chicago, Maywood, Illinois 60153 (113)
 Coordinate Regulation
of Translation by the PI 3-Kinase
and mTOR Pathways
Kathleen A. Martin and John Blenis
Department of Cell Biology, Harvard Medical School, Boston,
Massachusetts 02115

I. Overview of Translational Regulatory Pathways

A. The PI 3-Kinase Pathway
B. The mTOR Pathway
II. PI 3-K and mTOR Effectors which Regulate Translation
A. 5’ TOP mRNA and S6K1
B. Capped mRNA and eIF-4E
C. Other PI 3-K- and mTOR-Regulated Translation Initiation Factors
III. Regulation of Translational Effectors
A. 4E-BP1 Regulation
B. Regulation of S6K1
C. S6K2
IV. Coordinated Translational Control
A. Coordination of mRNA Splicing and Translation
B. Coordinated Growth and Proliferation in Liver Regeneration
C. PI 3-K, mTOR, Translation, and Cell Size
D. Coordination of the PI 3-K and mTOR Pathways
E. PI 3-K and mTOR Pathways in Cancer
V. Conclusions
References

Control of translation initiation is an important means by which cells tightly regulate

the critical processes of growth and proliferation. Multiple effector proteins contribute
to translation initiation of specially modified mRNAs that modulate these processes.
Coordinated regulation of these translational effectors by multiple signaling pathways
allows the integration of information regarding mitogenic signals, energy levels, and
nutrient sufficiency. The mTOR protein, in particular, serves as a sensor of all of these
signals and is thought to thus serve as a crucial checkpoint control protein. Signals from
the mTOR pathway converge with mitogenic inputs from the phosphoinositide (PI)
3-kinase pathway on translational effector proteins to coordinately control cellular
growth, size, and cell proliferation. The translational effectors regulated by the
PI 3-kinase and mTOR pathways and their roles in regulation of cellular growth will be
the primary focus of this review. C 2002, Elsevier Science (USA).

Advances in CANCER RESEARCH Copyright 2002, Elsevier Science (USA).

0065-230X/02 $35.00 1 All rights reserved.
2 Martin and Blenis

I. OVERVIEW OF TRANSLATIONAL
REGULATORY PATHWAYS

A. The PI 3-Kinase Pathway

Inputs from a wide range of extracellular signals converge on the phospho-

inositide 3-kinase (PI 3-K) family enzymes, which, in turn, regulate a host of
critical cellular processes, including proliferation, survival, motility, vesicle
trafficking, transcription, and protein synthesis (Fig. 1) (Rameh and Cantley,
1999). The role of this pathway in cell survival, in large part through reg-
ulation of the effector kinase Akt/PKB, is widely appreciated (Downward,
1998). The importance of this pathway in maintaining the balance between
proliferation, survival, and apoptosis is further underscored by the fact that
several effectors are protooncogenes, and the functional antagonist of this
pathway, PTEN, is a tumor suppressor gene frequently found to be mu-
tated in human tumors (Cantley and Neel, 1999). Notably, the PI 3-K path-
way coordinates the separable but related processes of cellular proliferation
(an increase in cell number) and growth (an increase in cell mass) in large
part through regulation of protein synthesis. In this review, we focus on the
role of PI 3-K targets in regulation of protein synthesis and cell growth.
Class I PI 3-Ks, including adapter (p85 family) and catalytic (p110
family) subunits, lie at the apex of an important growth factor stimulated
signaling pathway that governs many aspects of cell behavior. While PI
3-kinase enzymes catalyze both lipid and protein phosphorylation, the lipid

MITOGENS

PI 3-Kinase

phospholipid second PTEN

messengers

effector proteins

survival proliferation growth motility

Fig. 1 The PI 3-kinase pathway regulates multiple cellular functions in response to mitogenic
stimulation. Lipid second messengers generated by PI 3-K bind to and regulate multiple effector
proteins, which in turn modulate numerous critical processes in mammalian cells.
Coordinate Regulation of Translation 3

kinase-dependent functions are more thoroughly understood (Bondeva et al.,

1998). PI 3-Ks phosphorylate phosphatidylinositols at the 3 -OH position to
generate lipid second messengers that target a diverse array of downstream
effector proteins, allowing coordinated modulation of multiple cellular pro-
cesses. The major growth factor-induced PI 3-K-derived lipid messengers
are phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2) and phosphatidylinos-
itol 3,4,5-trisphosphate (PI-3,4,5P3) (Rameh and Cantley, 1999). Binding of
these lipids to effector proteins, especially those containing the plekstrin
homology (PH) domain motif, may induce conformational changes and/or
membrane targeting which alters their activities and access to other regula-
tory proteins or substrates. The PTEN lipid phosphatase dephosphorylates
PI 3-K-generated phospholipid second messengers, and thus coordinately in-
hibits the activation of the numerous downstream effectors of PI 3-K (Cantley
and Neel, 1999). The pharmacological inhibitors of PI 3-K, wortmannin and
LY294002, have been important tools for dissecting the roles of this pathway
and its effectors in vivo.

B. The mTOR Pathway

The lipid-sensitive PI 3-K effectors that regulate translation will be dis-

cussed in detail in this review. Many of these mediators are also regulated by
mTOR signaling, another pathway critical for translational control (Gingras
et al., 2001b). The integration of signals from the PI 3-K and mTOR path-
ways is an important emerging theme in the coordinated regulation of cell
growth and proliferation. A current model suggests that translational con-
trol is regulated by distinct, parallel signaling pathways that converge on
common effectors: mTOR senses nutrient sufficiency, energy levels, and per-
haps some mitogenic signals via phosphatidic acid (Fang et al., 2001) or
PI 3-K/Akt (Fig. 2) (Nave, 1999; Sekulic et al., 2000), while growth factor-
or mitogen-induced translation is mediated largely by the PI 3-K pathway,
with additional contributions from PKCs and MAPKs. Consistent with a
role in a nutrient checkpoint, inhibition of the mTOR pathway can over-
ride PI 3-K and other growth factor-derived signals to multiple transla-
tional effectors. Thus, no discussion of PI 3-K signaling would be complete
without an understanding of the inputs contributed by the mTOR pathway
(Fig. 3).
mTOR is the mammalian target of rapamycin (Sabers et al., 1995), also
known as FRAP (FKBP and rapamycin-associated protein) (Brown et al.,
1994), RAFT (rapamycin and FKBP12 target) (Sabatini et al., 1994), or
RAPT (Chiu et al., 1994). This protein is a serine/threonine protein kinase
that autophosphorylates and regulates exogenous substrates in translation
pathways, including S6Ks and 4E-BPs (to be discussed in detail in following
sections) (Brown et al., 1995; Brunn et al., 1997). mTOR signaling is
4 Martin and Blenis

ENERGY NUTRIENTS MITOGENS

(ATP) (amino acids) (phosphatidic acid)

amino acid
withdrawal

mTOR

TRANSLATION INITIATION

Fig. 2 Convergence of multiple upstream inputs on mTOR. Because mTOR integrates signals
from multiple stimuli, and because its inhibition suppresses the activity of translation initiation
effectors despite the presence of other mitogenic stimuli, mTOR can serve as an effective sensor
in checkpoint control of protein synthesis.

inhibited by a complex formed between the lipophilic macrolide anti-

biotic rapamycin (also known as Sirolimus) and the ubiquitous cellular
protein FKBP12 (FK506-binding protein 12 kDa) (Peterson et al., 2000).
Rapamycin inhibits multiple important functions of mammalian cells, in-
cluding protein synthesis, cell proliferation (Pyronnet and Sonenberg, 2001),

growth factors

ERK PI3K

nutrients

Rapamycin
mTOR

4E-BP S6K

S6
40S
eIF-4F

structured-mRNA 60S 5' TOP mRNA

Fig. 3 Coordinate regulation of translational effectors by growth factor and nutrient path-
ways. S6 kinases and 4E-BPs are regulated by multiple phosphorylations. Growth factor signals
are transduced to these effectors by the PI 3-K, ERK, and mTOR pathways, and nutrient/energy
sufficiency signals are mediated by the mTOR pathway. Both growth factor and nutrient/energy
signals are required for full activation of these translational effector proteins.
Coordinate Regulation of Translation 5

muscle hypertrophy (Rommel et al., 2001; Bodine et al., 2001), and cell
growth/cell size (Fingar, et al., 2002). While mTOR and FKBP12 are ubiq-
uitously expressed, the effects of rapamycin are more pronounced in cer-
tain cell types. Lymphocyte proliferation is highly sensitive to rapamycin,
which likely accounts for the drug’s utility as an immunosuppressant that re-
duces organ transplant rejection in clinical trials (Podbielski and Schoenberg,
2001).
mTOR contains a C-terminal domain with homology to the PI 3-K kinase
domain. It does not appear to be a functional lipid kinase, but is a member
of a growing family of proteins containing this homologous region known as
PIKKs, or phosphoinositide kinase-related kinases (Hoekstra, 1997). Many
members of the PIKK family are thought to serve checkpoint functions, in-
cluding ATM, ATR, and DNA-PK, which act as sensors for DNA damage and
repair (Hoekstra, 1997). A connection between DNA damage and mTOR-
dependent signaling has been observed (Tee and Proud, 2000), suggesting
that mTOR may also serve as a sensor of DNA damage. mTOR appears to
function in a nutritional checkpoint that senses amino acid availability. Phos-
phorylation of critical translational effectors of mTOR, including S6K1 and
4E-BP1, is sensitive to the availability of leucine and other branched chain
amino acids, and this effect is rapamycin-sensitive (Hara et al., 1998). Con-
versely, phosphorylation is inhibited by amino acid deprivation. Amino acid
signaling and rapamycin may employ similar mTOR regulatory mechanisms,
as a rapamycin-resistant mTOR mutant relieves the amino acid dependence
of S6K1 (Iiboshi et al., 1999). The mechanism by which leucine and other
amino acids signal to the mTOR pathway is not yet known but has been pos-
tulated to involve tRNA charging (Iiboshi et al., 1999), or leucine-stimulated
increased mitochondrial metabolism via oxidative decarboxylation and ac-
tivation of glutamate dehydrogenase (Xu et al., 2001; see Lynch, 2001,
for review). While amino acid-sensitive mTOR effectors are also subject to
PI 3-K regulation, a role for PI 3-K in amino acid signaling is unlikely, since
PI 3-K and Akt activities are unaffected by amino acid stimulation or depriva-
tion (Hara et al., 1998).
As protein synthesis is the most energetically expensive function performed
by the cell (Schmidt, 1999), integrated control of this process by a nutrient-
and energy-sensing checkpoint would be optimal. It has recently been sug-
gested that mTOR may indeed serve as a sensor of energy levels (Dennis
et al., 2001). Dennis et al. have shown that reduction of cellular ATP levels us-
ing the glycolytic inhibitor 2-deoxyglucose prevented insulin-induced activa-
tion of mTOR-regulated translational effectors. While all kinases require
ATP, the apparent Km for ATP for mTOR was estimated to be greater than
1 mM, as opposed to 10–20 μM for most other known kinases (Dennis
et al., 2001). This requirement for high, but physiological, concentrations
of ATP may reflect the ability of mTOR to physically sense cellular energy
6 Martin and Blenis

levels, as well as explain the technical challenges that have faced researchers
studying mTOR enzymatic activity in vitro.
Nutrient regulation of the yeast mTOR homologs, TOR1 and TOR2, has
been well documented (Rohde et al., 2001). The yeast TOR proteins have
provided many important insights into the roles and regulation of this path-
way in mammalian cells. While some mTOR functions may be mediated
through phosphorylation of targets, yeast models have suggested that TOR
1/2 may have more sweeping effects on multiple downstream cellular targets
through regulation of a phosphatase. In S. cerevisiae, TOR proteins stimulate
the association of the phosphatases Sit4 (PP6 homolog) and Pph21/22 (PP2A
homolog) with the regulatory protein Tap42. This association is inhibited by
nutrient insufficiency or rapamycin, and different mutations in Tap42 can
inhibit translation or confer rapamycin resistance, demonstrating the impor-
tance of this protein in TOR-mediated translational control (Di Como and
Arndt, 1996; Jiang and Broach, 1999). α4, the murine homolog of Tap42,
associates with the catalytic subunits of human phosphatases PP2A, PP6, or
PP4 (Murata et al., 1997; Chen et al., 1998). α4 binding alters PP2A substrate
specificity (Murata et al., 1997), and rapamycin inhibits the association of
α4 and PP2A (Murata et al., 1997), suggesting that an analogous pathway
may exist in mammals. Inhibition of S6K1 and 4E-BP1 by rapamycin may
be mediated via phosphatase activity, and PP2A has been shown to asso-
ciate with S6K1, but not with a rapamycin-resistant S6K1 mutant lacking
the N- and C-terminal regulatory domains (Fig. 4) (Peterson et al., 1999).

mTOR

Rapamycin or
α4
Amino acid withdrawal

α4
PP2A-C

PP2A-A PP2A-C

PP2A-B altered substrate specificity

S6K1 S6K1

(inactive) (active)

Fig. 4 Hypothetical model for mTOR regulation of phosphatase activity and translational
effectors in mammalian cells. Evidence from the TOR analogs in yeast, and from mammalian
cell experiments, suggests a model by which mTOR may modulate the activity and/or substrate
specificity of PP2A-family serine/threonine phosphatases by regulating binding of adapter sub-
units such as α4.
Coordinate Regulation of Translation 7

Activation of a phosphatase, as opposed to inhibition of a kinase, is an at-

tractive model to explain the rapid rapamycin-induced dephosphorylation
of at least 12 different sites on 4E-BP1 and S6K1 with dissimilar motifs
(Peterson et al., 1999). In this way, a nutrient-sensing checkpoint protein
could coordinately inhibit multiple diverse translational effectors when the
essential amino acid leucine is lacking, despite the continued presence of
positive growth factor signals (i.e., from the active PI 3-K pathway).
Recent work has suggested a novel mechanism by which serum mitogens
may also signal through mTOR to translational effectors. Serum-induced
phospholipase D (PLD) activity generates the second messenger phosphatidic
acid (PA), which binds to the FRB domain of mTOR (the same domain that
binds the FKBP12/rapamycin complex). This PA-mediated signaling stimu-
lates phosphorylation and activation of S6K1 and 4E-BP1 in a rapamycin-
and wortmannin-sensitive manner. Interestingly, PA does not alter the kinase
activity of mTOR. The authors suggest a model in which S6K1 and 4E-BP1
integrate nutrient signals through mTOR, as well as mitogenic signals via
PI 3-K and PA/mTOR (Fang et al., 2001). The roles and regulation of trans-
lational effector targets of both the mTOR and PI 3-K pathways will be
presented in detail below.

II. PI 3-K AND mTOR EFFECTORS WHICH

REGULATE TRANSLATION

Two downstream targets of PI 3-K and mTOR signaling, S6K1 and 4E-
BP1/eIF-4E, are major regulators of protein synthesis (Fig. 3). These effectors
will be introduced here, followed by a discussion of the intermediates that
transduce these signals to these targets.

A. 5’ TOP mRNA and S6K1

Translation initiation rates for different mRNAs can vary dramatically

depending on the degree and type of secondary structure present in the 5 un-
translated region of the message. One modification, the 5 terminal oligopy-
rimidine tract (5 TOP), a stretch of 4–14 pyrimidine bases at the extreme 5
end of the message, marks a particular subset of mRNAs for inefficient trans-
lation initiation under basal conditions (Meyuhas, 2000). Insulin or other
growth factor stimulation rapidly induces translation of these messages in a
rapamycin-sensitive manner (Jefferies et al., 1994). These 5 TOP mRNAs
typically encode components of the protein synthetic machinery itself, in-
cluding ribosomal proteins and translation elongation factors. Induction
8 Martin and Blenis

of 5 TOP mRNA translation upregulates ribosome biogenesis and overall

translational capacity (Meyuhas, 2000).
Translation initiation of 5 TOP mRNAs is thought to be mediated by
the 40S ribosomal protein S6. Growth factor-stimulated phosphorylation of
S6 at multiple C-terminal residues correlates with translation initiation of
these 5 TOP mRNAs (Jefferies et al., 1997). S6 is phosphorylated by the
70-kDa S6 kinase 1 (p70 S6K1) (Kozma et al., 1990), a ubiquitously ex-
pressed mitogen- and amino acid-sensitive protein kinase. S6K1 activation
and subsequent S6 phosphorylation is a conserved mitogenic response, as all
mitogenic stimuli, including growth factors, protooncogene products, phor-
bol esters, and cytokines induce S6K1 activity (Dufner and Thomas, 1999).
Enhanced ribosome biogenesis and translational capacity is a conserved res-
ponse to growth signals.
Recent data have questioned whether S6K1 regulates 5 TOP-mediated
mRNA translation through S6 phosphorylation, suggesting that this process
is instead dependent on PI 3-K and mTOR signaling, but not on S6K1 or
the ribosomal S6 protein (Tang et al., 2001). Although many aspects of
this study contradict the published work of others (Jefferies et al., 1997;
Lee-Fruman et al., 1999), this and other studies suggest the likely possibility
that other as yet unidentified targets for the S6 kinases exist that are impor-
tant in mediating their physiological function (Shima et al., 1998; Montagne
et al., 1999). Notably, mice lacking S6K1 exhibit a small-animal phenotype
despite normal phosphorylation of S6 and 5 TOP translation (likely due to
redundant function by S6K2) (Shima et al., 1998), thus other S6K1 targets
may contribute to regulation of animal size. Whether or not ribosomal S6
phosphorylation modulates 5 TOP mRNA translation, it is likely an essen-
tial component of a proliferation checkpoint mechanism as revealed by con-
ditional S6 deletion studies (Volarevic et al., 2000) (see Section IV.B).

B. Capped mRNA and eIF-4E

A methyl cap structure (m7GpppN) is found at the 5 end of all mRNAs

transcribed in the nucleus. Interaction of this cap with translation initiation
factor eIF-4E (eukaryotic initiation factor-4E) is an important step in loading
these messages on the ribosome (Sonenberg and Gingras, 1998). The cap-
binding subunit eIF-4E is present in rate limiting quantities relative to other
components of the translational apparatus (Rau et al., 1996), and thus serves
as a key regulatory translation factor. Notably, eIF-4E provides an additional
degree of translational control toward mRNAs containing a high degree of
secondary structure in the 5 UTR (Rousseau et al., 1996). These stable secon-
dary structures include hairpin loops and upstream AUGs, modifications
which mark mRNAs encoding proteins important for cell growth and cell
Coordinate Regulation of Translation 9

Fig. 5 Regulation of eIF-4F formation by 4E-BP1. eIF-4E is sequestered by hypophosphory-

lated 4E-BP1. Phosphorylation of 4E-BP1 by growth factor- and nutrient-sensing signaling
pathways allows release of eIF-4E, which subsequently associates with the scaffolding protein
eIF-4G and helicase eIF-4A to form the eIF-4F complex.

cycle progression, including growth factors, receptors, cyclins, and signaling

proteins (Sonenberg and Gingras, 1998). Efficient translation initiation of
these highly structured mRNAs requires unwinding by a helicase, eIF-4A.
eIF-4E links these messages to the helicase by binding both the mRNA cap
and a scaffolding protein, eIF-4G, which also binds eIF-4A. This complex
of eIF-4E, eIF-4G, and eIF-4A is referred to collectively as eIF-4F, and is
important for recruiting the 40S ribosome to the message (Fig. 5) (Sonenberg
and Gingras, 1998).
Like 5 TOP mRNAs, eIF-4E-dependent growth regulatory messages are
poorly translated in quiescent cells, and translation of these structured
mRNAs is similarly upregulated following growth factor stimulation. In
quiescent cells, eIF-4E is sequestered by the 4E-BP (eIF-4E-binding protein)
family repressor proteins (Pause et al., 1994) (4E-BP1 is also known as
PHAS-I; phosphorylated, heat- and acid-stable-Insulin responsive (Lin et al.,
1994)). In their hypophosphorylated state, 4E-BPs bind eIF-4E in a manner
mutually exclusive with eIF-4G, a scaffolding subunit of the eIF-4F initiation
complex. This complex binds capped mRNAs (Haghighat, 1995; Marcotri-
giano et al., 1997). 4EB-P1 binding to eIF-4E inhibits formation of eIF-4F
complexes. This inhibition is relieved when 4E-BPs are phosphorylated
in response to growth factors and dissociate from eIF-4E (Sonenberg and
Gingras, 1998). In addition to regulation by 4E-BPs, phosphorylation of
eIF-4E itself by the ERK effectors Mnk1/2 is thought to promote translation
initiation (Waskiewicz et al., 1997; Scheper et al., 2001). Phosphorylation of
eIF-4E alone, however, is not sufficient for eIF-4F assembly (Herbert et al.,
2000). 4E-BP1, the best characterized 4E-BP, is regulated by signals from
10 Martin and Blenis

the PI-3K, ERK, and mTOR pathways, and these inputs will be discussed in
detail below.

C. Other PI 3-K- and mTOR-Regulated Translation

Initiation Factors

Initiation factor eIF-4G also likely integrates PI 3-K and mTOR signals.
It undergoes serum- or insulin-stimulated phosphorylation on at least three
sites that are sensitive to wortmannin and rapamycin, but the upstream ki-
nases are not yet known (Raught et al., 2000). The effects on eIF-4G function
are also unknown, but it has been postulated that such phosphorylations may
alter its structure, which may modulate its scaffold function during eIF-4F
assembly.
Another translational regulator responsive to both PI 3-K and mTOR sig-
nals is eIF-4B. This RNA binding protein may function as a link between ribo-
somal and messenger RNAs, and stimulates eIF-4A helicase activity (Rozen
et al., 1990). eIF-4B is phosphorylated in response to growth factors in a
rapamycin- and wortmannin-sensitive manner, and is a substrate for S6K1
in vitro (Morley and Traugh, 1993). The effect of phosphorylation is not
known, but it is likely that regulation of eIF-4B may have the most profound
effect on translation of mRNAs with highly structured 5 UTRs.
A translational effector that appears to be regulated by the PI 3-K but
not by the mTOR or MAPK pathways is eIF-2B. This guanine nucleotide
exchange factor is a component of eIF2, which catalyzes binding of the
initiator Met-tRNA to the ribosome, an important step in initiation (Price
and Proud, 1994). The eIF-2Bε subunit is inhibited when phosphorylated by
GSK3 (Welsh et al., 1998). Insulin relieves this suppression through an Akt-
induced inhibition of GSK3 (Cross et al., 1995; Takata et al., 1999). Thus,
the PI 3-K pathway regulates translation through multiple effectors of the
initiation apparatus, including S6K1, 4E-BP1, eIF-4G, eIF-4B, and eIF-2B.

III. REGULATION OF TRANSLATIONAL EFFECTORS

A. 4E-BP1 Regulation

4E-BPs are the major factors regulating eIF-4E activity, and thus, sub-
sequent formation of the eIF-4F initiation complex. Hypophosphorylated
4E-BP1 binds eIF-4E with high affinity (Pause et al., 1994; Lin et al., 1994).
The 4E-BP1–eIF-4E interaction is disrupted following sequential 4E-BP1
Coordinate Regulation of Translation 11

Fig. 6 Regulation of 4E-BP1 phosphorylation. Sequential phosphorylation of 4E-BP1 is medi-

ated by nutrient- and growth factor-sensing signaling pathways. Phosphorylation of the priming
sites Thr37 and Thr46 precedes phosphorylation of Thr70 and Ser65, to allow release of eIF-4E.

phosphorylation at multiple sites. Thr37 and Thr46 are basally phosphory-

lated and are substrates for phosphorylation by mTOR in vitro (Fig. 6)
(Gingras et al., 1999; Mothe-Satney et al., 2000a). 4E-BP1 undergoes or-
dered, sequential phosphorylation, with Thr37 and Thr46 serving as pre-
requisite priming sites for serum-induced phosphorylation at Thr70 and
Ser65 (Gingras et al., 1999, 2001a; Mothe-Satney et al., 2000b). Studies
with phosphospecific antibodies suggest that Ser65 may be the final site to
be phosphorylated (Mothe-Satney et al., 2000a; Gingras et al., 2001a). This
site is also likely critical for disruption of the eIF-4E interaction, as the eIF-4E
binding site in 4E-BP1 is flanked by Thr46 and Ser65 (Haghighat, 1995),
and phosphorylation of Ser65 prevents this association in vitro. Due to the
multiple phosphorylations, 4E-BP1 from stimulated cells migrates as a triplet
of bands in SDS-polyacrylamide gels. Only the most slowly migrating band
reacts with phospho-Ser65, and this species fails to copurify with eIF-4E
(Mothe-Satney et al., 2000a).
Like S6K1, Ser65 and Thr70 are regulated by both PI 3-K and mTOR
pathways, as they are sensitive to wortmannin and rapamycin (Gingras
et al., 1998; Mothe-Satney et al., 2000a). S6K1 is not an in vivo 4E-BP1
kinase, but S6K1 and 4E-BP1 are thought to function in parallel pathways
which bifurcate downstream of mTOR. S6K1 and 4E-BP1 may share a com-
mon rapamycin-sensitive activator, since overexpression of S6K1 can inhibit
4E-BP1 phosphorylation (von Manteuffel et al., 1997). One report states that
Ser65 and Thr70 are also phosphorylated by mTOR in vitro, but only when
12 Martin and Blenis

mTOR is incubated with an “activating” antibody, mTAb1 (Mothe-Satney

et al., 2000a). Alternately, dissociation of eIF-4E from 4E-BP1 may involve
4E-BP1 phosphorylation by an mTOR-associated kinase (Heesom and
Denton, 1999). Another model to explain 4E-BP1 inactivation is that these
sites are phosphorylated by PI 3-K-regulated kinases, and dephosphorylated
by PP2A-type phosphatases, which are activated following mTOR inhibition
(Peterson et al., 1999). Akt appears to be an important regulator of 4E-BP1
in vivo, as expression of a constitutively active mutant induces 4E-BP1 phos-
phorylation, while a dominant negative is inhibitory (Gingras et al., 1998;
Dufner et al., 1999; Takata et al., 1999). Akt may regulate a 4E-BP1 kinase,
as Akt itself does not directly phosphorylate 4E-BP1 in vitro (Gingras et al.,
1998).
It has recently been shown that the mTOR-regulated nPKCδ (Parekh et al.,
1999) can phosphorylate 4E-BP1 in a rapamycin-sensitive manner, although
the target site has not yet been identified (Kumar et al., 2000a). Through
an inhibitory association with mTOR, the c-abl tyrosine kinase is a neg-
ative regulator of 4E-BP1 function in response to DNA damage (Kumar
et al., 2000b). Finally, 4E-BP1 phosphorylation may occur by a phorbol
ester-stimulated, PI 3-K independent mechanism which may be mediated by
the MEK/ERK cascade (Herbert et al., 2000, 2002). Basal MEK activity
is also required for insulin-stimulated 4E-BP1 phosphorylation and eIF-4F
assembly cascade (Herbert et al., 2000). ERK2 can phosphorylate 4E-BP1
in vitro (Fadden et al., 1997) and in vivo in vascular smooth muscle cells
(Rao et al., 1999).
Other 4E-BP family members, including 4E-BP2 and 4E-BP3 (Poulin et al.,
1998) have been identified. These are related to 4E-BP1, with the greatest
sequence conservation in the eIF-4E-binding motif (Gingras et al., 2001b).
These proteins seem to serve a similar function and are likewise regulated
by phosphorylation. 4E-BP1 and −2, but not 4E-BP3, contain a four-amino
acid motif (Arg-Ala-Ile-Pro “RAIP”) in the N-terminus that is required for
efficient phosphorylation (Tee and Proud, 2002). Early studies with 4E-BP2
suggest that slight differences in regulation may lead to different kinetics
of eIF-4E dissociation (Gingras et al., 2001b; Grolleau, 1999). Thus, the
function of these isoforms may allow for tissue-specific differences in growth
factor stimulated translational responses (Grolleau, 1999).

B. Regulation of S6K1

The PI 3-K and mTOR pathways are major signaling pathways regulating
S6K1 activity (Chung et al., 1992, 1994). The S6K1 (and 4E-BP1) require-
ment for inputs from both the PI 3-K and mTOR pathways, as well as by
other mitogen activated signaling pathways, suggests a mechanism by which
Coordinate Regulation of Translation 13

cells can integrate nutrient capacity and growth factor stimulated protein
synthesis.
S6K1 exists as two isoforms. The 70-kDa αII isoform is largely cytosolic
in localization. An 85-kDa αI isoform is identical to p70 with the exception
of an additional 23 amino acids at the N-terminus encoding a nuclear lo-
calization signal (Coffer and Woodgett, 1991; Reinhard et al., 1994). The
function of nuclear S6K1 is unclear, but S6K1 can phosphorylate the trans-
cription factor cremτ , suggesting a possible role in transcriptional control
(de Groot et al., 1994). The two S6K1 isoforms appear to be regulated
similarly in all systems examined, except for slightly delayed kinetics of p85
activation relative to p70 in response to pressure overload in cardiomyocytes
(Laser et al., 1998).
S6K1 kinase activity is regulated by at least nine growth factor-induced
phosphorylation events. The first of these, targeting multiple sites in the
C-terminus, is thought to relieve autoinhibition by intramolecular interac-
tions (Cheatham et al., 1995; Weng et al., 1995). The kinase domain at
the core of the molecule is flanked by N- and C-terminal regulatory do-
mains (Fig. 7). A model of S6K1 activation based on structure/function
mutagenesis studies suggests that acidic amino acids in the N-terminus in-
teract with basic residues in the C-terminus to stabilize an autoinhibitory
inactive conformation. In this inactive state, a pseudosubstrate region in
the C-terminus with high homology to ribosomal S6 occludes the kinase

Fig. 7 Structure of S6 kinases. Shown is a schematic of the primary structures of the shorter
isoforms of S6K1 (αII) and S6K2 (βII). Mitogen-stimulated phosphorylation sites are indicated
by amino acid number. The activation loop (T229/228 TFCGT), linker region (S371/370 SPDD)
and hydrophobic motif (T389/388 FLGFTY) sites are perfectly conserved between S6K1 and
S6K2, but there is some divergence in the proline-directed C-terminal sites. S6K2 exhibits regions
of divergence from S6K1 in the N- and C-terminal regulatory domains, including the presence
of a C-terminal polyproline-rich domain and nuclear localization signal.
14 Martin and Blenis

domain (Cheatham et al., 1995; Weng et al., 1995). An early step in S6K1
activation is mitogen-induced phosphorylation of C-terminal regulatory sites
(Ser404, Ser411, Ser418, Thr421, Ser424), which disrupt this interaction,
perhaps mediated by ERK or p38 MAP kinases (Weng et al., 1998;
A. Romanelli, unpublished results). Phospho-specific antibodies reveal that
these sites are also sensitive to inhibition by rapamycin or wortmannin (Weng
et al., 1998). Phosphorylation of sites in an internal regulatory domain and
the kinase domain follow, including Ser371, Thr389, and Thr229. Ser371
phosphorylation is essential for S6K1 activity, and is mediated by an as yet
undetermined mechanism but is insensitive to rapamycin and wortmannin
(Moser et al., 1997; A. Romanelli, unpublished observations). Thr229, lo-
cated in the catalytic activation loop, is essential for kinase activity and
sensitive to wortmannin and rapamycin (Weng et al., 1998). This site is
phosphorylated by phosphoinositide-dependent kinase 1 (PDK1), a con-
stitutively active kinase whose subcellular localization and access to sub-
strates is regulated by PI 3-K-derived phospholipids (Williams et al., 2000;
Alessi et al., 1998; Pullen et al., 1998). The importance of Thr229 is high-
lighted by the fact that an inhibitor of PDK1 signaling, n-alpha-tosyl-l-
phenylalanyl chloromethyl ketone (TPCK), is a potent inhibitor of S6K1
activity (Grammer and Blenis, 1996; Ballif et al., 2001). Further, mutation
of this site (T229A) abolishes S6K1 activity and subsequent Thr389 phos-
phorylation (Weng et al., 1998).
Phosphorylation of Thr389 in the regulatory domain appears to be central
to S6K1 activation, as it is exquisitely sensitive to inhibition by rapamycin,
and thought to be the final and rate-limiting step in S6K1 activation (Weng
et al., 1998; A. Romanelli, unpublished observations). While the importance
of this amino acid in S6K1 function is universally accepted, the mechanism
underlying Thr389 regulation is currently controversial. This site has been
reported to be directly phosphorylated by mTOR in vitro (Burnett et al.,
1998a). However, Thr389 is still phosphorylated in a mitogen-sensitive man-
ner in a truncation mutant of S6K1 that is rapamycin resistant, indicating that
mitogens can stimulate Thr389 phosphorylation independently of mTOR
(S. Schalm, unpublished observations). The NIMA family kinases NEK6/7
have been shown to phosphorylate Thr389 in vivo and in vitro (Belham
et al., 2001). Activity of these kinases, however, is only weakly stimulated
by insulin and partially sensitive to wortmannin, while Thr389 phospho-
rylation is entirely wortmannin sensitive, suggesting that other mechanisms
may contribute to Thr389 regulation.
Regulation of the critical Thr389 site is also dependent on PDK1, as IGF-I
fails to induce Thr389 phosphorylation in PDK1 null cells (Williams et al.,
2000), and TPCK inhibits phosphorylation of both Thr229 and Thr389
(Ballif et al., 2001). It is also possible that S6K1 autophosphorylation
Coordinate Regulation of Translation 15

contributes to the phosphorylation state of Thr389, as we have observed

that optimal and prolonged Thr389 phosphorylation does not occur in a
kinase inactive S6K1 (K100R) point mutant (A. Romanelli, unpublished ob-
servations). This mechanism is consistent with the requirement for PDK1
phosphorylation of the catalytic activation loop site (Thr229). Finally, the
many mitogen-stimulated phosphorylation sites in S6K1, including Thr389,
are rapidly dephosphorylated after rapamycin treatment. As discussed ear-
lier, an mTOR-regulated phosphatase is a possible mechanism which could
rapidly inhibit many sites regulated by diverse upstream kinases (Peterson
et al., 1999).
A highly conserved sequence in the N-terminus of S6K1 and S6K2 (amino
acids 5 to 9 of S6K1) has recently been revealed to be a critical TOR signaling
(TOS) motif that is essential for mTOR-dependent signaling. The TOS do-
main mediates mTOR regulation of two distinct regions of S6K1: this motif
is required for phosphorylation of Thr389, as well as for release of a negative
regulatory mechanism mediated by the S6K1 C-terminus. The importance
of the TOS motif in mTOR signaling is underscored by its identification in
other mTOR-regulated proteins. This motif is conserved throughout evo-
lution in the C-termini of 4E-BPs. Mutation of the TOS motif in 4E-BP1
similarly blocks its mitogen-dependent phosphorylation (Schalm and Blenis,
2002).
It has been long known that conventional PKCs (cPKCs) can also con-
tribute to S6K1 activation (Blenis and Erikson, 1986), but the precise molecu-
lar mechanism is still not well documented. PDGF receptor mutants deficient
in PLCγ binding and subsequent cPKC activation are impaired in signaling
to S6K1, while mutants lacking the PI 3-K binding site suggest that the
PI 3-K pathway is the major contributor to S6K1 activation (Chung et al.,
1994). Separable PI 3-K and cPKC inputs were also documented in B cells
(Monfar et al., 1995). Similarly, S6K1 activation by B cell antigen receptor
cross-linking is dependent on both PI 3-K and cPKC-dependent pathways
and involves the tyrosine kinase Syk (Li et al., 1999).

1. SUBCELLULAR LOCALIZATION AND REGULATION OF S6Ks

S6K1 is activated by multiple PI 3-K effectors, including Cdc42, Rac (Chou
and Blenis, 1996), PDK1 (Alessi et al., 1998; Pullen et al., 1998), and atypi-
cal PKCζ (Romanelli et al., 1999) and PKCλ (Fig. 8) (Akimoto et al., 1998).
While PDK1 directly phosphorylates S6K1 (Alessi et al., 1998; Pullen et al.,
1998), the mechanism of activation by these other effectors is an ongoing
subject of investigation. The low-molecular-weight G proteins Cdc42 and
Rac activate S6K1 independent of the p38 or JNK pathways (Chou and
Blenis, 1996). These G proteins, however, can bind to S6K1 in vivo in a
16 Martin and Blenis

mTOR
PI3Kp110 PI3K p85

PP2A?
Cdc42/Rac PKCζ PDK1 Akt

S6K1

Fig. 8 Regulation of S6K1 by PI 3-K and mTOR. The PI 3-K effectors Cdc42, Rac, PKCζ ,
PDK1, and Akt have all been implicated in regulation of S6K1. Only PDK1 is known to directly
phosphorylate S6K1 (at Thr229). Evidence suggests that mTOR may directly phosphorylate
S6K1 at Thr389, and/or may regulate all S6K1 phosphorylation sites through regulation of a
PP2A-type phosphatase. Multiple PI 3-K effectors exist in a complex with S6K1, and the PI 3-K
p85 adapter subunit may mediate association of mTOR and S6K1.

rapamycin- and wortmannin-insensitive manner. This association is required

for Cdc42 activation of S6K1. Isoprenylation of the G protein is also required
(Chou and Blenis, 1996). These data suggest that interaction of these G pro-
teins may activate S6K1 by inducing a conformational change in the kinase,
and/or by targeting the kinase to a membrane, bringing it in proximity with
other membrane-localized activators including PDK1, aPKCs, and Akt. We
have also reported that S6K1 coimmunoprecipitates with PDK1 or PKCζ ,
and that PDK1 and PKCζ can associate with each other (Romanelli et al.,
1999). Constitutively active mutants of Akt support a membrane targeting
model, as only those which localize to the plasma membrane are capable
of activating S6K1 (Dufner et al., 1999). These combined data suggest that
S6K1 may participate in a complex of PI 3-K-regulated effector proteins
which facilitates signaling in this pathway. The existence of such scaffolding
mechanisms has been suggested for other pathways, including MAPK path-
ways (Kholodenko et al., 2000; Garrington and Johnson, 1999; Burack and
Shaw, 2000). It is not known whether a common central scaffolding molecule
exists in this PI 3-K model, but other evidence suggests that these interac-
tions may take place at the plasma membrane and/or cytoskeleton. Finally,
coprecipitation of S6K1, mTOR, and the p85 subunit of PI 3-K suggest that
a ternary (or larger) complex integrates S6K1 activation by the PI 3-K and
mTOR pathways (Gonzalez-Garcia et al., 2002) (see Section IV.D for further
detail).
Coordinate Regulation of Translation 17

The S6K1 activators Cdc42 and Rac are also important regulators of the
cytoskeleton that mediate many structural changes contributing to cell motil-
ity (Erickson and Cerione, 2001). Several studies suggest that S6K1 might
also play a role in motility, as it has been shown to colocalize with stress
fibers, and that thrombin-induced elongation and organization of stress fibers
is rapamycin sensitive in fibroblasts (Crouch, 1997). Furthermore, S6K1
colocalizes with actin arc structures at the leading edge of migrating cells
(Berven and Crouch, 2000). Interestingly, several factors known to regulate
and associate with S6K1, the atypical PKCs ζ or λ (Romanelli et al., 1999;
Akimoto et al., 1998) and Cdc42 (Chou and Blenis, 1996), have recently
been reported to associate with each other in a GTP-dependent manner
(Coghlan et al., 2000). This study demonstrated that activated Cdc42 in-
duced stress fiber loss, and that this process required active aPKCs. A role
for S6K1 in this process was not addressed, but these data further document
the clustering of similarly regulated signaling proteins at cytoskeletal struc-
tures.
Two-hybrid and biochemical analyses identified the F-actin binding pro-
tein neurabin as a binding partner for S6K1 in neural cells (Burnett et al.,
1998b). A PDZ domain in this neural-specific cytoskeletal-associated pro-
tein binds to the extreme C-terminus of S6K1 in a serum- and rapamycin-
independent manner. The mRNAs for neurabin and S6K1 colocalize in var-
ious brain structures, including the hippocampus and cerebellum, and it
has been suggested that these proteins colocalize at nerve terminals. Co-
expression of S6K1 and neurabin in nonneural tissues leads to a modest
induction of S6K1 activity (Burnett et al., 1998b), supporting the model
that “scaffold”-mediated targeting of S6K1 to cellular locales enriched in
regulatory molecules may facilitate S6K1 activation.
Subcellular localization is important for regulation of the mTOR path-
way, as well as for PI 3-K effectors, and perhaps especially so for common
effectors of both pathways such as S6Ks. The ubiquitously expressed pro-
tein gephyrin serves to cluster glycine receptors at postsynaptic nerve termi-
nals, and has been identified as a binding partner for mTOR (Sabatini et al.,
1999). mTOR interaction with gephyrin mediates its subcellular localization
and is essential for its ability to regulate S6K1 and 4E-BP1. Furthermore,
an intriguing study has suggested that nuclear shuttling of mTOR may be
necessary for mitogen-stimulated activation of translational effectors, as in-
hibition of nuclear export using leptomycin B inhibited phosphorylation of
both S6K1 and 4E-BP1 (Kim and Chen, 2000). Such nuclear/cytoplasmic
shuttling of mTOR suggests a possible regulatory mechanism for primarily
nuclear isoforms such as p85-S6K1 or the S6K2 proteins. It is likely that
future studies will further clarify the mechanisms by which components of
signaling pathways are brought together to function efficiently and specifi-
cally.
18 Martin and Blenis

C. S6K2

For many years it was thought that S6K1 was the only in vivo S6 kinase.
However, several groups recently identified a homolog closely related to
S6K1, now called S6K2 (also called p70β, SRK) (Shima et al., 1998; Gout
et al., 1998; Lee-Fruman et al., 1999; Koh et al., 1999). In addition to
isolation based on database searches for novel proteins with homology to
S6K1 (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al., 1999; Koh
et al., 1999), S6K2 was identified when normal S6 phosphorylation and 5
TOP mRNA translation was discovered in cells derived from mice lacking
both p70 and p85 isoforms of S6K1 (Shima et al., 1998). As the mRNA for
S6K2 was found to be upregulated in these mice, it is likely that S6K2 may
supply the S6 phosphorylation function in vivo in the absence of S6K1. Using
a rapamycin-resistant S6K2 mutant, we find that S6K2 is indeed an in vivo
S6 kinase, as S6 phosphorylation persists in rapamycin-treated cells stably
overexpressing this mutant, when S6K1 and other mTOR-effectors are maxi-
mally inhibited (K. Martin, unpublished observations). Whether additional
in vivo S6 kinases might also exist remains to be determined.
Mice lacking S6K1 through homozygous deletion demonstrate a small-
animal phenotype despite normal S6 phosphorylation and 5 TOP mRNA
translation (Shima et al., 1998), suggesting that S6 regulation is not the crit-
ical determinant of animal size, but that S6K1 may mediate other important
nonredundant functions that contribute to size regulation. Because, unlike
S6K1, both S6K2 isoforms encode a common C-terminal nuclear localization
signal (Koh et al., 1999), it is likely that S6K2 mediates unique nuclear func-
tions. Several structural features and differential regulation further suggest
that S6K2 mediates distinct functions. While S6K2 is highly homologous
to S6K1 overall, there are regions of divergence in both the amino- and
carboxyl-terminal regulatory regions (Fig. 7). In addition to the unique C-
terminal NLS, S6K2 contains a C-terminal polyproline-rich region absent in
S6K1. The role of the polyproline domain is not yet known, but deletion
of this domain reportedly does not affect wortmannin or rapamycin sensi-
tivity (Gout et al., 1998). Chimeras swapping the region of the polyproline
domain between S6K1 and S6K2 failed to reveal an obvious function for
this domain (S. Schalm, unpublished observations). S6K2 is activated by the
same stimuli that activate S6K1, and is potently inhibited by wortmannin
and rapamycin (Shima et al., 1998; Gout et al., 1998; Lee-Fruman et al.,
1999; Koh et al., 1999), suggesting that it also functions as an effector of
the PI 3-K and mTOR pathways.
Regulation of S6K2 is largely similar to S6K1, with some intriguing differ-
ences, likely arising due to sequence variations and/or differential subcellular
localization. Despite the primarily nuclear expression of S6K2, it is regulated
Coordinate Regulation of Translation 19

by the PI 3-K effectors Cdc42, Rac, PDK1, PKCζ , (Martin et al., 2001a), and
Akt (Koh et al., 1999). One difference in S6K regulation was that atypical
PKCζ was a more potent activator of S6K2. Interestingly, point mutation
which destroys the S6K2 nuclear localization signal modestly potentiates its
activation by PI 3-K effectors (Martin et al., 2001a). This regulation by cy-
tosolic effectors suggests that S6K2 may exit the nucleus during the course
of activation.
Like S6K1, S6K2 exists as two alternately spliced isoforms, which differ
by an additional N-terminal 13 amino acids present in S6K2β1 but lacking
in S6K2β2 (Gout et al., 1998). Although the C-terminal nuclear localiza-
tion signal is common to both S6K2 isoforms (Koh et al., 1999), it has been
suggested that the unique basic sequence at the N-terminus of S6K2β1 con-
tributes to its nuclear localization as well, as overexpressed S6K2β1 is found
primarily in the nucleus, while overexpressed S6K2β2 can be found in both
the nucleus and the cytosol (Minami et al., 2001).
It has been suggested that S6K2 is less sensitive to rapamycin or wort-
mannin than is S6K1 (Gout et al., 1998; Minami et al., 2001). It is likely,
however, that this reflects a difference in inactivation, as opposed to initial
activation. Minami et al. observed that S6K2 overexpressed in cells contin-
ually cultured in 10% serum was less sensitive to inhibition by addition of
rapamycin or wortmannin to the medium. We and others, however, have
found that activation of S6K2 in serum-starved cells by the addition of in-
sulin or serum is nearly completely inhibited by rapamycin or wortmannin
(Lee-Fruman et al., 1999; Koh et al., 1999).
The most notable functional difference between S6K1 and S6K2 is the role
of the C-terminal autoinhibitory domain. Deletion of this regulatory region,
which lies just C-terminal to the kinase domain, has a modest inhibitory
effect on S6K1, yet has a potent stimulatory effect on S6K2 that is dramati-
cally enhanced by coexpression of PI 3-K effectors, such as Cdc42 or PDK1
(Martin et al., 2001b). These data suggest that this domain participates in
potent repression of S6K2 kinase activity. Furthermore, this repression of
S6K2 may be relieved by inputs from the MEK/ERK1/2 pathway, as full-
length S6K2 is highly sensitive to inhibition by the MEK inhibitor U0126,
while S6K1 is far less sensitive to MEK inhibition (Martin et al., 2001b).
The S6K2 U0126 sensitivity was evident for EGF, a potent ERK agonist, but
not for insulin, a poor ERK agonist in the HEK293 cells used. Activation
of S6K2 by G protein-coupled receptor agonists in cardiomyocytes was also
found to be highly MEK dependent (Wang et al., 2001). The MEK pathway
has been implicated in regulation of the C-terminal phosphorylation sites in
both S6K1 and S6K2 (Lenormand et al., 1996; Scott and Lawrence, 1997;
Mukhopadhyay et al., 1992; Herbert et al., 2000; A. Romanelli, unpub-
lished results), but may play a more critical role in the initial steps of S6K2
20 Martin and Blenis

activation. It is also possible that the presence of the polyproline-rich domain

in proximity to the C-terminal regulatory phosphorylation sites may confer
differential S6K2 regulation.
While S6K1 contains a motif at its C-terminus which may allow its reg-
ulation by PDZ domain proteins such as neurabin (Burnett et al., 1998b),
S6K2 lacks such a motif. The absence of a C-terminal PDZ-binding sequence
may account for some differences in S6K2 regulation by this domain (Martin
et al., 2001b). However, both S6K1 and S6K2 are regulated by isoprenylated
Cdc42 and Rac (Chou and Blenis, 1996; Martin et al., 2001a), suggesting
that membrane association may be important in regulation of both kinases.

IV. COORDINATED TRANSLATIONAL CONTROL

A. Coordination of mRNA Splicing and Translation

Recent work on regulation of mRNA splicing by the low-molecular-weight

G protein Cdc42 suggests that the PI 3-K and mTOR pathways may coordi-
nate the related processes of mRNA splicing and translation, in part, through
S6K1 (Wilson et al., 2000). Cdc42 has been shown to be an effector of the
PI 3-K pathway, and an activator of both S6K1 and S6K2 (Chou and Blenis,
1996; Martin et al., 2001a). Activated Cdc42 stimulates binding of the cap-
binding complex (CBC) to nuclear capped mRNAs (Wilson et al., 1999)
and stimulates pre-mRNA splicing independent of its PAK, ACK, or WASP
effector functions (Wilson et al., 2000). Notably, splicing stimulated by an
activated Cdc42 mutant is sensitive to rapamycin, but not to wortmannin.
Signaling from Cdc42 to S6K1 has been proposed to mediate this effect,
as S6K1 was found to phosphorylate the CBC subunit CBP80 in vitro at
growth factor-stimulated rapamycin-sensitive sites. It is likely that PI 3-K
lies upstream in this pathway, but that the downstream constitutively acti-
vated Cdc42 mutant employed is resistant to the effects of wortmannin, as
overexpression of p70 S6K1 and constitutively active PI 3-K enhances splic-
ing in the presence of endogenous Cdc42. The S6K1 phosphorylation sites
flank a nuclear localization signal, but ablation or acidic substitution of the
sites does not alter nuclear localization of CBC. S6K1 phosphorylates CBP80
in vitro, but it has not yet been determined whether the cytosolic p70 or nu-
clear p85 S6K1, or even the nuclear S6K2 isoforms, may mediate this event
in vivo. One model suggests that the nuclear CBC escorts the capped mRNA
complex from the nucleus to the cytosol, where it binds to the eIF-4E cap-
binding translation initiation factor (Wilson et al., 2000; Visa et al., 1996).
Thus, Cdc42/S6K inputs may coordinate mRNA splicing with PI 3-K- and
mTOR-regulated translation initiation. Cdc42 has been implicated in cell
Coordinate Regulation of Translation 21

transformation (Lin et al., 1997). Interestingly, an activated Cdc42 double

mutant deficient in splicing regulation is also deficient in transformation,
suggesting that this new S6K-mediated function of Cdc42 may contribute to
the transformed phenotype (Wilson et al., 2000).

B. Coordinated Growth and Proliferation

in Liver Regeneration

p85 alpha PI 3-K knockout mice reveal that these enzymes play an essential
role in the liver (Fruman et al., 2000). Liver regeneration following partial
hepatectomy provides a model system for assessing the intertwined processes
of cell growth and proliferation. Partial hepatectomy increases circulating
levels of hepatocyte growth factors, and markedly induces the activities of
PI 3-K, Akt, and S6K1 (Michalopoulos and DeFrances, 1997; Hong et al.,
2000; Jiang et al., 2001), and expression of a novel liver-specific PI 3-K,
termed PI 3-KIIγ (Ono et al., 1998). Mice with diminished levels (CRE/lox
conditional knockout) of ribosomal S6 protein in liver are impaired in re-
covery from partial hepatectomy (Volarevic et al., 2000). Hepatocytes from
these livers exhibit abnormal ribosome profiles and progress through early
G1 phase, as indicated by normal induction of cyclin D. The cells do not
progress to S phase, as cyclins E and A mRNA and protein are lacking. The
authors propose that this arrest may be due to activation of a checkpoint
which senses abnormal ribosome biogenesis. This study raises interesting
questions regarding the coordination of protein synthesis and proliferation.
Interpretation of these studies is complicated, however, by the long half-life
of the S6 protein. Because S6 protein was present at even 5 days after CRE
induction, it should be noted that the data were observed in the presence
of reduced levels, but not complete lack, of S6. A complete knockout of
S6 protein, if viable, may have a more immediate and potent effect on liver
regeneration following both starvation and hepatectomy.
Another interesting finding from the partial hepatectomy model is that
4E-BP1 is not required for rapamycin-sensitive liver regeneration. Partial
hepatectomy induces S6K1 activation and 4E-BP1 phosphorylation and sub-
sequent reduction in eIF-4E binding and repression (Jiang et al., 2001).
Normal animals treated with rapamycin suffer impaired liver regeneration
(Francavilla et al., 1992; Jiang et al., 2001). It was found that under these
conditions, S6K1 phosphorylation and activity is markedly inhibited, while
4E-BP1 phosphorylation persists, and eIF-4E cap-binding activity conse-
quently increases in hepatocytes from rapamycin-treated rats after partial
hepatectomy (Jiang et al., 2001). These data suggest not only an mTOR-
independent phosphorylation of 4E-BP1, but also that S6K1 may be an
essential target in rapamycin-sensitive regeneration in vivo. The role of the
22 Martin and Blenis

rapamycin-sensitive related kinase S6K2 in hepatocyte models has not yet

been addressed, but this kinase may be another candidate for regulation of
protein synthesis and proliferation in regenerating liver. These findings dif-
fer from reports of rapamycin-sensitive 4E-BP1 phosphorylation in vitro,
and suggest that the in vivo environment following hepatectomy is not mir-
rored in tissue culture experiments. The Erk pathway has been implicated in
4E-BP phosphorylation in other cell types (Fadden et al., 1997; Rao et al.,
1999), but Erk1/2, p38, or Jnk activation was not detected following hepa-
tectomy (Jiang et al., 2001). Other rapamycin-insensitive effector kinases in
the PI 3-K or PKC pathways known to regulate 4E-BPs in other systems
may be responsible for the rapamycin-insensitive phosphorylation following
hepatectomy. In light of these studies suggesting the importance of S6K and
S6 in liver regeneration, it will be of interest to determine the effect of partial
hepatectomy in S6K1 knockout mice. Embryonic fibroblasts from these mice
are reported to have normal S6 phosphorylation, perhaps due to compensa-
tion by S6K2 (Shima et al., 1998). Whether S6 phosphorylation is entirely
normal in adult hepatocytes in vivo, however, has not yet been established.
This may shed light on the relative roles of S6K1 versus S6K2 in the dy-
namic liver model. Comparison of individual and combined knockouts of
S6K1 and S6K2 would be the best tool to address this question.

C. PI 3-K, mTOR, Translation, and Cell Size

The critical role of PI 3-K in cell growth and proliferation is underscored

by the conservation of this pathway from C. elegans to humans. This conser-
vation has allowed for elegant genetic studies in multiple organisms, which
have revealed the importance of the PI 3-K pathway and specific effectors
in control of cell size. Mutations of the Drosophila insulin receptor (INR)
(Chen et al., 1996), IRS homolog (chico) (Bohni et al., 1999), PI 3-K (dp110)
(Leevers et al., 1996), Akt (dAkt) (Verdu et al., 1999), TOR/FRAP (dTOR)
(Zhang et al., 2000; Oldham et al., 2000), or S6K1 (dS6K) (Montagne et al.,
1999) give rise to a small cell phenotype. Similarly, overexpression of a
d4EBP mutant having high affinity for deIF4E reduces Drosophila cell size
(Miron et al., 2001). In the dS6K mutant flies, cell number is preserved, but
each individual cell is smaller in size than wild-type cells, producing nor-
mally proportioned animals with a 50% reduction in body size (Montagne
et al., 1999). These data suggest that dS6K regulates cell size and, subse-
quently, organ and body size, but does not influence patterning, cell-fate, or
spatial decisions. S6-dependent control of ribosome biogenesis and, transla-
tional capacity is an attractive hypothesis to explain these phenotypes, but
it is more likely that other targets of S6K1 are involved in cell size control:
Homozygous deletion of S6K1 in mice results in a small animal phenotype
Coordinate Regulation of Translation 23

despite the presence of normal S6 phosphorylation and 5 TOP mRNA reg-

ulation, presumably mediated by the intact function of the homolog S6K2
(Shima et al., 1998). These mice have pancreatic β-cells of reduced size, ren-
dering them hypoinsulinemic (Pende et al., 2000). Notably, the dS6K mutant
phenotype is far more severe in flies, where it includes female sterility, devel-
opmental delay, and early death (Montagne et al., 1999). The S6K1-/- mice
are viable, fertile, and only 20% smaller than wild type (Shima et al., 1998).
This may reflect the role played by S6K2 in mammalian cells (Shima et al.,
1998), while there is thought to be only one S6K species in Drosophila.
The PI 3-K pathway has also been implicated in cell size regulation in a
cardiac hypertrophy model, where the variable of cell proliferation is elim-
inated due to the terminally differentiated status of cardiomyocytes (Shioi
et al., 2000). Cardiac-specific expression of activated or dominant negative
PI 3-K resulted in transgenic mice with larger or smaller hearts, respectively,
with corresponding regulation of Akt and S6K1. Similarly, overexpression
of active PTEN inhibited, and catalytically inactive PTEN promoted, car-
diomyocyte hypertrophy (Schwartzbauer and Robbins, 2001).
Genetic studies demonstrate that upstream PI 3-K components may play
more diverse roles in regulation of both growth and proliferation. While
chico and dS6K mutant flies are viable, deletions of INR, PI 3-K, or dAkt
result in embryonic lethality (Chen et al., 1996; Leevers et al., 1996; Verdu
et al., 1999). Mutations in INR, chico, PI 3-K, and dAkt affect cell num-
ber as well as cell size (Leevers, 1999). dPTEN has been shown to act as a
tumor suppressor in flies, reducing cell number and cell size, by antagoniz-
ing the PI 3-K pathway (Goberdhan et al., 1999). Other evidence, however,
points to a role for S6K1 in proliferation as well as translational control and
cell size. A rapamycin-resistant S6K1 mutant rescued rapamycin-sensitive
inhibition of E2F transcriptional responses in lymphocytes (Brennan et al.,
1999). Others suggest that S6K1 is not essential for proliferation after ob-
servation of rapamycin-sensitive S6K1 activity in cells whose proliferation
is rapamycin resistant (Hosoi et al., 1998; Louro et al., 1999; Slavik et al.,
2001). We propose that there may be multiple downstream targets of the
mTOR pathway that may confer rapamycin resistance to proliferation. A
lesion in the pathway downstream of S6K1 does not necessarily exclude a
role for S6K1 in proliferation.
While genetic data from Drosophila and in vivo mouse models have be-
gun to identify signaling molecules that function to regulate cell growth
and cell size, the biochemical signaling pathways that cooperate to con-
trol cell growth in mammalian cells are less well understood. Treatment
of asynchronously cycling cultured mammalian cells with rapamycin or
LY294002 reduces cell size as well as cell cycle progression and prolifer-
ation, implicating a role for mTOR and PI3-K in control of mammalian
cell size (Fingar et al., 2002). It is the inhibition of mTOR that mediates
24 Martin and Blenis

rapamycin’s effect on cell size, as expression of a rapamycinresistant mutant

of mTOR rescues the rapamycin-induced small cell size phenotype. Similarly,
treatment of differentiated C2C12 myotubes or isolated rat skeletal muscle
with rapamycin blocks muscle hypertrophy (Bodine et al., 2001; Rommel
et al., 2001). Consistent with a role for mTOR and PI3-K in control of cell
size, overexpression of either S6K1 or eIF4E increases cell size, while coex-
pression of both proteins additively increases cell size, demonstrating that
the mTOR to S6K1 and mTOR to 4EBP/eIF4E pathways function in par-
allel downstream of mTOR to coordinately regulate cell size (Fingar et al.,
2002). Similarly, overexpression of S6K1 or Akt1 in differentiated C2C12
myotubes induces muscle cell hypertrophy (Rommel et al., 2001). Consis-
tent with the reduction in cell size observed upon overexpression of a d4EBP
mutant with high affinity for deIF4E in Drosophila (Miron et al., 2001),
overexpression of a phosphorylation site mutant of 4EBP1 (Thr37/46Ala)
in cultured mammalian cells also reduces cell size (Fingar et al., 2002). Fur-
ther evidence supporting a role for mTOR-mediated signaling in control of
mammalian cell size is that an S6K1 phosphorylation site acidic substitution
mutant (E389D3E) that exhibits partial rapamycin resistance, or overexpres-
sion of eIF4E, partially rescues the reduced cell size phenotype induced by
rapamycin (Fingar et al., 2002). Lastly, while cell cycle progression and cell
growth are normally tightly coordinated during cellular proliferation (cells
tend to remain fairly constant in size through multiple cell division cycles),
the two processes can be experimentally dissociated in mammalian cells
(Fingar et al., 2002), confirming what Lee Hartwell and colleagues first ob-
served in budding yeast 25 years ago (Johnston et al., 1977). When cell
cycle progression is blocked upon expression of cell cycle inhibitory proteins
(such as the cdk inhibitors p16 or p21, or dominant-negative cdk2), cells
continue to grow to increased size, indicating that cell cycle progression and
cell growth are separable and distinct processes. Importantly, this growth to
increased cell size is blocked by rapamycin or LY294002. The mechanisms
that allow tight coordination of cell cycle progression and cell growth are
poorly understood (see Section IV.D). Data from numerous experimental
systems all point to an important role for signaling molecules that regulate
protein translation as important regulators of cell growth and cell size.

D. Coordination of the PI 3-K and mTOR Pathways

Two recent genetic studies of mutations in the Drosophila TOR (dTOR)

protein elegantly assessed the relationship between the PI 3-K and TOR
pathways (Oldham et al., 2000; Zhang et al., 2000). Both studies conclude
that the dTOR pathway serves a nutrient checkpoint function that converges
Coordinate Regulation of Translation 25

on growth factor-regulated translational effectors of the PI 3-K pathway.

Disruption of dTOR function prevents flies from developing past the larval
stage (Zhang et al., 2000). The cellular phenotypes perfectly mimic the effects
of amino acid starvation (Oldham et al., 2000). As with mutations in the
PI 3-K pathway, including dS6K (Montagne et al., 1999), loss of dTOR
function results in a cell autonomous cell size defect, resulting in reductions
in organ and body size (Zhang et al., 2000; Oldham et al., 2000). In dTOR
mutants, proliferation rates are also reduced, characterized by an increased
number of cells in G1 phase, and fewer in S and G2 (Zhang et al., 2000).
Cyclin E overexpression rescued the G1 arrest, and cyclin E protein levels
were greatly reduced in dTOR mutant flies, suggesting that this cyclin is a
critical downstream target of TOR regulation (Zhang et al., 2000).
The dTOR mutants provide important observations on the role and reg-
ulation of dS6K. Oldham et al. observed diminished S6 phosphorylation,
but an upregulation of dS6K protein levels in dTOR mutants or following
amino acid deprivation, suggesting dTOR-mediated feedback regulation of
dS6K. Interestingly, mutations in the PI 3-K pathway do not affect dS6K
levels (Oldham et al., 2000). The role of dS6K as an essential downstream
target of dTOR was emphasized by Zhang et al. (2000), who demonstrated
that overexpression of dS6K could rescue development of flies with dimin-
ished dTOR function, allowing them to develop to adulthood. The dS6K-
rescued flies were fertile and developed normally, but were slightly reduced
in size, suggesting that while dS6K is a major in vivo effector of the TOR
pathway, other dTOR-mediated functions may be necessary. Consistent with
this hypothesis, constitutively activated dS6K could not rescue the most se-
vere dTOR mutants to adulthood, but did allow progression to the pupal
stage (Zhang et al., 2000). Oldham et al. also report a failure of dS6K to
rescue severe dTOR mutants. Thus, a low level of dTOR activity is neces-
sary to supply functions of dTOR effectors in addition to dS6K. One such
function may be eIF-4E activation, as eIF-4E mutants exhibit a growth ar-
rest phenotype (Zhang et al., 2000). However, eIF-4E overexpression alone
was not sufficient to rescue the dTOR mutant phenotype (Zhang et al.,
2000).
The common effectors of both PI 3-K and mTOR have lead some to hy-
pothesize that these proteins may function in a linear signaling pathway. The
dTOR studies in Drosophila, along with other evidence, suggest that this is
not the case. dTOR mutations complement mutations in PTEN, suggesting
that dTOR affects downstream components in the PI 3-K pathway (Oldham
et al., 2000; Zhang et al., 2000). Mutations in dTOR are more severe than
those in PI 3-K, however, as dTOR mutants arrest at an earlier larval stage
than those of the Drosophila PI 3-K subunits Dp110 or Dp60 (Weinkove
et al., 1999; Zhang et al., 2000). In contrast to dTOR mutants, PI 3-K
26 Martin and Blenis

mutants fail to upregulate dS6K, and exhibit dissimilar larval phenotypes

(Oldham et al., 2000). Thus, the Drosophila genetic data are inconsistent
with a role for dTOR as a downstream effector of PI 3-K.
Studies in mammalian cells also support the model that PI 3-K and mTOR
regulate independent but parallel pathways that converge on common effec-
tors. While the autokinase activity of mTOR is indeed wortmannin sensi-
tive, this inhibition requires a concentration 100-fold greater than the dose
that effectively inhibits PI 3-K activity (Brunn et al., 1996). Other stud-
ies have suggested that the PI 3-K effector Akt phosphorylates mTOR in a
putative negative regulatory domain (Ser2448) (Nave et al., 1999; Sekulic
et al., 2000). Mutation of this site, however, does not inhibit mTOR acti-
vation of S6K1 (Nave, 1999; Sekulic et al., 2000), and, notably, this site
is not conserved in dTOR, despite high sequence conservation between
dTOR and mTOR in other regions, including the kinase and HEAT do-
mains (Zhang et al., 2000). In addition, growth factor activation of the PI
3-K pathway and Akt induces little to no change in mTOR kinase activ-
ity (Scott et al., 1998; Sekulic et al., 2000). Notably, S6K1 is activated by
constitutively active Akt mutants only when these mutants are targeted to
the plasma membrane (Dufner et al., 1999), and dominant negative Akt in-
hibits S6K1 (Takehashi, et al., 2002). This Akt regulation of S6K1 may be
explained by its inhibition of TSC2 (Manning, unpublished observations,
see Section IV.E.). S6K1 itself suggests separable mTOR and PI 3-K path-
ways, as an S6K1 truncation mutant is rapamycin resistant, yet retains sensi-
tivity to wortmannin (Cheatham et al., 1995; Weng et al., 1995). Genetic
studies, however, suggest that the PI3K and mTOR pathways may also func-
tion linearly, as rapamycin analogs inhibit malignancies induced by lesions
in PI3K/PTEN signaling (Podsypanina et al., 2001; Neshat et al., 2001; Aoki
et al., 2001) (see Section IV.E).
The mTOR gene has been mutated, but not deleted, in mice. This study
reveals mTOR is essential for embryonic development (Hentges et al., 2001).
Mutation of mTOR or treatment of embryos with rapamycin results in a “flat
top” embryonic lethal phenotype, characterized by lack of the telencephalon.
Interestingly, cells from the mutant mouse exhibited a defect in proliferation,
but not in cell size. It should be noted, however, that S6K1 and 4E-BP1
phosphorylation and activities were reduced, but not absent, and may have
been sufficient to maintain cell size regulation. This study suggests, however,
that mTOR is an important mediator of mitogenic signaling in the developing
mouse.
The issue of the relationship between PI 3-K and mTOR remains contro-
versial, as experimental evidence is difficult to interpret for several reasons.
Much of the controversy revolves around the ability of the PI 3-K path-
way, and specifically, Akt, to regulate mTOR kinase activity. Measurement
Coordinate Regulation of Translation 27

of mTOR kinase activity is technically challenging and may account for

disparate results from different groups. A larger issue, however, is the mis-
leading assumption that mTOR kinase activity always mediates mTOR func-
tion. For example, PA activation of mTOR substrates is dependent upon PA
binding to mTOR, but stimulation or inhibition of PLD/PA signaling does
not alter mTOR kinase activity (Fang et al., 2001). There is evidence for
direct phosphorylation of S6K1 and 4E-BP1 by mTOR in vitro, but there is
also compelling evidence that mTOR-mediated phosphatase regulation may
be more important in regulating translational effectors. Equally plausible is
involvement of both mechanisms. Finally, whether or not PI 3-K can signal
to mTOR is difficult to address by pharmacologic means, because rapamycin
completely overrides all other signals that regulate mTOR effectors.
Much of the data regarding PI 3-K and mTOR can be explained by two
possible models. One model suggests that PI 3-K and mTOR function in
distinct and parallel signaling pathways, converging upon common down-
stream effectors. Inputs from both pathways are required, as inhibition of
either pathway is sufficient to override incoming signals from the other. This
model is consistent with a checkpoint function for mTOR. Another model
would suggest that in addition to functioning independently in parallel, PI
3-K-derived signals may also contribute to activation of mTOR, creating a
linear pathway from PI 3-K to mTOR. Because of the technical concerns
described, it is difficult to discriminate between these views.
Finally, a physical basis by which parallel PI 3-K and mTOR inputs con-
verge upon activation of S6K1 has been proposed in a recent study, which
suggests that the p85 adapter subunit of PI 3-K nucleates a complex between
S6K1 and mTOR (Gonzalez-Garcia et al., 2002). A p85 truncation mutant
(p65PI3K) which lacks the C-terminal SH2 domain was shown to retain the
ability to activate the p110 PI 3-K catalytic subunit and Akt. However,
this mutant failed to promote serum-stimulated activation of S6K1. While
p85PI3K forms a complex with S6K1 and mTOR, p65PI3K does so inefficiently.
Interestingly, a rapamycin-resistant S6K1 truncation mutant can be activated
by either p65PI3K or p85PI3K, suggesting that the crucial function of the PI 3-K
regulatory subunit is recruitment of mTOR. This study suggests a model by
which PI 3-K is necessary for (1) phosphorylation of S6K1 at Thr229 and
Thr389 by PI 3-K-regulated kinases, and (2) recruitment of mTOR to an
S6K1-containing complex, which confers protection from mTOR-regulated
phosphatases (Peterson et al., 1999). This model suggests a physical basis
by which the PI 3-K and mTOR pathways integrate growth factor- and
nutrient-derived signals at the level of the translational effector S6K1. It will
be interesting to determine whether this complex formation is mediated by
the TOS domain of S6K1, whether this regulation applies to activation of
primarily nuclear S6 kinases (p85 S6K1 and S6K2 isoforms), and whether
28 Martin and Blenis

PI 3-K and mTOR signals similarly converge in a physical complex on 4E-BP

factors.

E. PI 3-K and mTOR Pathways in Cancer

The importance of these signaling pathways in regulating cell growth and

proliferation is underscored by the presence of mutations in multiple com-
ponents of these pathways in human cancers. Gain of function mutations in
PI 3-Kp110, Akt, mTOR, and eIF-4E, and loss of PTEN function lesions
have been detected in multiple human cancers (Vogt, 2001). Germline muta-
tion of PTEN has been implicated as the lesion in Cowden’s disease, a human
genetic disorder characterized by multiple hamartomas and susceptibility to
multiple benign and malignant tumors (Marsh et al., 1999). Elevated S6K
activity has been observed in uterine tumors from mice heterozygous for
PTEN, and treatment with the rapamycin ester CCI 779, an analog designed
for intravenous delivery, reduced tumor size and proliferation (Podsypanina
et al., 2001). The growth and proliferation of tumor cell lines lacking PTEN
were found to be more sensitive to CCI 779 than PTEN+/+ cells (Neshat
et al., 2001). These PTEN null cells also exhibited increased S6K1 activity
and 4E-BP1 phosphorylation. Interestingly, oncogenic transformation of
chick embryo fibroblasts by PI 3-K or Akt gain of function could be in-
hibited by rapamycin (Aoki et al., 2001). This study found that mTOR was
essential for PI 3-K-induced oncogenesis, but not for transformation induced
by oncogenes that function in other signaling pathways. These studies sug-
gest that rapamycin may be an effective anticancer agent, particularly for
tumors arising due to lesions in the PI 3-K pathway.
The tuberous sclerosis complex (TSC) genes have been recently identified
as tumor suppressors that negatively regulate both cell growth and prolifer-
ation. These genes are responsible for a human inherited disorder character-
ized by development of benign hamartomatous tumors in multiple organs,
and predisposition to malignant tumor formation (Jones et al., 1997). Loss
of function of TSC1 or 2 results in benign tumors characterized by large
cells. Genetic analyses in Drosophila suggest that the TSC1/2 complex lies
on a parallel pathway that inhibits insulin signaling downstream of Akt
(Gao and Pan, 2001). Interestingly, genetic epistasis experiments place dS6K
downstream of TSC1/2 and demonstrate that dS6K and TSC1/2 serve an-
tagonistic functions (Fig. 9) (Potter et al., 2001; Tapon et al., 2001). This
also appears to be true in mammalian cells, as S6K1 activity is upregulated
in mouse cells lacking TSC1 (Kwiatkowski et al., 2002) and in human cells
lacking TSC2 (Goncharova et al., 2002). Akt phosphorylation and inhibi-
tion of the TSC2 gene product tuberin suggests a mechanism by which Akt
contributes to S6K1 activation (Manning, unpublished observations), and
Coordinate Regulation of Translation 29

Fig. 9 The tuberous sclerosis complex (TSC) opposes insulin signaling. The TSC1/2 genes
have been identified as negative regulators of S6K function and cell growth. The PI 3-K effectors
Akt, PDK1, and PKCζ contribute to S6K activation. Akt relieves TSC inhibition by phospho-
rylation and inhibition of the TSC2 gene product tuberin. It is not yet known whether tuberin
inhibits S6K1 directly or via mTOR.

may explain S6K1 inhibition by dominant negative Akt (Takehashi et al.,

2002). These examples from the cancer literature further highlight the in-
terdependence of the PI 3-K and mTOR pathways, and the contribution of
their effectors to growth and proliferation.

V. CONCLUSIONS

The PI 3-K pathway mediates many essential cellular functions, including

survival, proliferation, and cell growth/cell size. We have described PI 3-K
effectors which transduce signals to regulate translation initiation, ribo-
some biogenesis, and translational capacity. These include the S6 kinases,
4E-BPs and eIF-4E, eIF-4G, and eIF-4B. These PI 3-K effectors also integrate
signals from other growth factor-stimulated pathways, including conven-
tional PKCs and MAPKs. Importantly, these effectors integrate the incom-
ing growth factor signals with mitogenic inputs from the mTOR pathway,
a crucial nutrient- and energy-sensing checkpoint pathway, ensuring ade-
quate amino acid resources to meet the demand for new protein synthesis.
Common subcellular localization of effectors and regulatory molecules may
facilitate such integrated signal transduction. The influence of growth factor-
and nutrient-sensitive pathways on growth and proliferation is a continu-
ing subject of investigation that will likely reveal important new insights
into normal physiology and pathological conditions including hypertrophy,
diabetes, and cancer.
30 Martin and Blenis

ACKNOWLEDGMENTS

The authors thank Diane Fingar, Angela Romanelli, Stefanie Schalm, Celeste Richardson,
and Andrew Tee for their contributions to this manuscript.

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This Page Intentionally Left Blank
 Histone Acetyltransferases and
Deacetylases in the Control of Cell
Proliferation and Differentiation
Heike Lehrmann, Linda Louise Pritchard,
and Annick Harel-Bellan
CNRS UPR 9079, Institut André Lwoff, 94800 Villejuif, France

I. Introduction
II. Acetylation of Histones
III. Histone Acetyltransferases
A. GNAT-Family (Gcn5-Related N-acetyltransferases)
B. MYST-Family
C. CBP/p300 Family
IV. Histone Deacetylases and Cell Cycle Regulation
A. Mad /Max and HDACs
B. Rb and HDACs
C. HDACs and Cancer
V. Muscle Differentiation
A. Interaction of MyoD with CBP/p300 and PCAF
B. MyoD and HDAC1
C. MEF2 and Deacetylases
VI. Hematopoiesis
A. GATA-1
B. EKLF
C. CBP/p300 and Hematopoietic Disorders
VII. Huntington’s Disease
VIII. Histone Acetylation in Combination with Other
Chromatin Modifications
IX. Conclusion
References

Histone acetylation and deacetylation are chromatin-modifying processes that have

fundamental importance for transcriptional regulation. Transcriptionally active chro-
matin regions show a high degree of histone acetylation, whereas deacetylation events
are generally linked to transcriptional silencing. Many of the acetylating and deacety-
lating enzymes were originally identified as transcriptional coactivators or repressors.
Their histone-modifying enzymatic activity was discovered more recently, opening up a
whole new area of research. Histone acetyltransferases such as CREB-binding protein

Advances in CANCER RESEARCH Copyright 2002, Elsevier Science (USA).

0065-230X/02 $35.00 41 All rights reserved.
Other documents randomly have
different content
He was forty-two and by the standards of the time a 48
rich man. Since his income was sufficient for his needs,
he made up his mind to retire. A fellow printer named
David Hall took over the management of his printing
shop. Franklin moved to a quiet part of town, at Race
and Second streets, and bought a 300-acre farm in
Burlington, New Jersey, where he could practice the art
of a gentleman farmer.

It was time, he believed, to devote the remaining years

of his life to his friends, to his writing, to the pursuit of
learning. Particularly a branch of learning that had
occupied his attention on and off for the past several
years—the study of electricity.

49
 5
THE THUNDER GIANT

A few years before his retirement, Franklin, on a visit to

Boston, attended a display of electrical tricks given by a
Dr. Adam Spencer of Scotland. There is no record of the
nature of these “electrical tricks.” Franklin commented
later that Dr. Spencer was no expert and that they were
imperfectly performed. Since he had never seen
anything of the sort before, he was “surpris’d and
pleased.”

That sparks could be produced by friction had been

known since ancient times. Little more was known
about electricity until, in the first part of the eighteenth
century, a young Frenchman, Charles François du Fay,
identified two different types of electricity: vitreous,
produced by rubbing glass with silk; resinous, produced
by rubbing resin with wool or fur. Such frictional
electricity was brief-lived. Sparks flashed and were
gone, and that was the end of it.

Was there any way in which electric charges could be 50

preserved from the rapid decay which they underwent
in the air? Around 1747 two scientists were working
independently on this problem—E. C. von Kleist of
Pomerania and Pieter van Musschenbroek of the
University of Leyden. Within a few months of each
other, they had found a method of storing electricity in a
container. The Leyden jar, this container was named. It
was the first electrical condenser.

In one experiment Musschenbroek suspended a glass

phial of water from a gun barrel by a wire which went
down through a cork in the phial a few inches into the
water. The gun barrel, hanging on a silk rope, had a
metallic fringe inserted into the barrel which touched an
electrically charged glass globe. A friend who was
watching him, a man named Cunaeus, happened to
grasp the phial with one hand and the wire with
another. Immediately he felt a strange and startling
sensation—reportedly the first manmade electric shock
in history.

Musschenbroek repeated what Cunaeus had done, this

time using a small glass bowl as his “Leyden jar.” “I
would not take a second shock for the King of France,”
he said.

Van Kleist in Pomerania produced the same effect. He

lined the inside and outside of his Leyden jar with silver
foil, charged the inner coat heavily, connected it with
the outer foil by a wire which he held in his hand—and
felt a violent shock run into his arm and chest.

A Leyden jar could take any number of forms. Even a 51

wine bottle would serve. The type used most frequently
during the next few years was a glass tube, some two
and a half feet long, and just big enough around so that
a man might grasp it easily in his hand. The advantage
of this size and shape was that it could most
conveniently be electrified, which was then done by
hand, by rubbing the glass with a cloth or buckskin. This
simple device gave impetus to research on electricity
throughout Europe. It also provided a new form of
entertainment.

Performers went from town to town with their Leyden

jars, giving spectators the thrill of receiving electric
shocks, and extolling the marvels of “electrical fire.”
Louis XV of France invited his guests to watch a novel
spectacle arranged by his court philosopher, Abbé Jean-
Antoine Nollet. The King’s Guard in full uniform lined up
before the throne, holding hands. The first one was
instructed to grasp the wire or chain connected to the
Leyden jar. They all jumped convulsively into the air as
an electric current passed through them.

In Italy some scientists tried to cure paralysis by electric

shock, claiming moderate success. In May 1748, for
instance, Jean-François Calgagnia, thirty-five years old,
was given an electric shock from a simple cylinder-type
Leyden jar. Since the age of twelve, his left arm had
been so paralyzed he could not lift his hand to his head.
After the first electrical treatment he at once raised his
arm and touched his face. There is no record as to
whether the cure was permanent.

After Franklin became aware of this phenomenon, he

was agog to try experiments on his own. He wrote of
his interest to a London friend, Peter Collinson, a
Quaker merchant and member of the Royal Society.
Collinson promptly sent him a glass tube, along with
suggestions as to how it might be used for electrical
experiments. This was all Franklin needed to get
started.

He was not trained in scientific matters as were many of 52

his European contemporaries. He was unfamiliar with
scientific jargon, and could only write about what he
was doing in everyday language. But he had those
qualities that are innate in any scientist, with or without
a university degree—an inquiring mind, patience, and
persistence.

His experiments, beginning with the winter of 1746,

covered a wide range. He melted brass and steel
needles by electricity, magnetized needles, fired dry
gunpowder by an electric spark. He stripped the gilding
from a book, and he electrified a small metallic crown
above an engraving of the King of England—so that
whoever touched the crown received a shock!

His home was soon so crowded with curious visitors

trooping up and down the stairs, he could hardly get
any work done. He solved the problem by having a
glass blower make tubes similar to his, passing them
out to friends so they could make their own
experiments.

Several of the Junto members worked closely with him.

At first they electrified the tube, as was still done in
Europe, by vigorously rubbing one side of it with a piece
of buckskin. One of the club members, a Silversmith
named Philip Synge, devised a sort of grindstone, which
revolved the tube as one turned a handle. To charge the
tube with electricity, all that was needed was to hold the
buckskin against the glass as it revolved, a vast saving
in physical labor.

Another invention of Franklin and his associates was the

first storage battery. For electrical plates they used
eleven window glass panes about six by eight inches in
size, covered with sheets of lead, and hung on silk cords
by means of hooks of lead wire. They found it as easy
to charge this “battery” with frictional electricity as to
charge a single pane of glass.

Among his disciples was an unemployed Baptist minister 53

named Ebenezer Kinnersley. Franklin suggested he
might both serve science and earn his living if he held
electrical demonstrations. Kinnersley’s first
announcement of a lecture, held in Newport, described
“electrical fire” as having “an appearance like fishes
swimming in the air,” claiming this fire would “live in
water, a river not being sufficient to quench the smallest
spark of it.” He promised his audience such wonders as
“electrified money, which scarce anybody will take when
offered ... a curious machine acting by means of electric
fire, and playing a variety of tunes on eight musical bells
... the force of the electric spark, making a fair hole
through a quire of paper....”

Kinnersley lectured in the colonies and the West Indies

and was hugely successful. Neither he nor any of the
other collaborators could rival Franklin’s own
achievements.

Early in 1747, he gave the names of positive and

negative (or plus and minus) to the two types of
electricity, to replace the unwieldy terms, resinous and
vitreous. Positive and negative electricity became part of
the scientific vocabulary. He was the first to refer to the
conductivity of certain substances. Electricity passed
easily through metals and water; they were conductive.
Glass and wood were nonconductive, unless they were
wet. He also noted that pointed metal rods were
wonderfully effective “in drawing off and throwing off
the electrical fire.”
After he retired in 1748, he spent much more time on
electricity. To Peter Collinson in London he wrote, “I
never was before engaged in any study that so totally
engrossed my attention and my time as this has lately
done.” He kept Collinson informed in detail of his
experiments, not because he thought he had the final
word but in the hope that his experiments might
possibly prove helpful to English scientists.

It was to Collinson he described an electrical party to be 54

held on the banks of the Schuylkill River in the spring of
1749: “A turkey is to be killed for our dinner by the
electrical shock, and roasted by the electrical jack,
before a fire kindled by the electrified bottle; when the
healths of all the famous electricians in England,
Holland, France, and Germany are to be drank in
electrified bumpers, under the discharge of guns from
an electrical battery.”

For Christmas dinner that year, he started to electrocute

another turkey, but inadvertently gave himself the shock
intended for the fowl: “The company present ... say that
the flash was very great and the crack as loud as a
pistol.... I neither saw the one nor heard the other.... I
then felt ... a universal blow throughout my whole body
from head to foot.... That part of my hand and fingers
which held the chain was left white, as though the
blood had been driven out, and remained so eight or
ten minutes after, feeling like dead flesh; and I had a
numbness in my arms and the back of my neck which
continued till the next morning but wore off.”

He was apologetic rather than frightened by the near

catastrophe, comparing himself to the Irishman “who,
being about to steal powder, made a hole in the cask
with a hot iron.”
This was soon after he had come to the conclusion that
what he now called “electrical fluid” had much in
common with lightning—that indeed they might be one
and the same thing. He was not the first to propose this
theory but no one before him had been able to suggest
how it might be tested.

Thunder and lightning had mystified humanity since the 55

beginning of recorded history. The Greeks had held that
thunderbolts were launched by the god Jupiter. (One
Greek philosopher, Empedocles, thought that lightning
was caused by the rays of the sun striking the clouds.)
Hunters of primitive tribes prayed to the god of
lightning, who was a killer, as they wished to be. Certain
medicine men were said to be endowed with the gift of
summoning lightning at will.

Since biblical days, lightning was assumed to be an act

of heavenly vengeance, but no one could explain the
paradox that it struck church steeples more frequently
than other buildings. In medieval times, people believed
that ringing church bells would keep lightning away, a
belief that survived the death of countless unfortunate
bell ringers.

About 1718, an English scientist, Jonathan Edwards,

suggested that thunder and lightning might be
produced by a “mighty fermentation, that is some way
promoted by the cool moisture, and perhaps attraction
of the clouds.” There had been very few other attempts
to give a scientific explanation of the phenomenon, and
even in Franklin’s time many preachers considered
lightning a manifestation of the Divine Will.

“Electrical fluid” and lightning had in common, Franklin

wrote in his notes on November 7, 1749, that they both
gave light, had a crooked direction and swift motion,
and were conducted by metals. Both melted metals and
could destroy animals. Since they were similar in so
many respects, would it not follow that lightning, like
“electrical fluid” would be attracted by pointed rods?
“Let the experiment be made.”

By May 1750, he was sure enough of his hypothesis that

he elaborated to Peter Collinson the advantages to
humanity of what later were called lightning rods:

I am of the opinion that houses, ships, and even

towers and churches may be effectually secured
from the strokes of lightning ... if, instead of the
round balls of wood or metal which are commonly
placed on tops of weathercocks, vanes, or masts,
there should be a rod of iron eight or ten feet in
length, sharpened gradually to a point like a needle
... the electric fire would, I think, be drawn out of a
cloud silently, before it could come near enough to
strike....

Did he guess that he was on the verge of the most 56

momentous discovery of the century—one which would
assure his name a place among the immortals? It is
fairly certain he was more interested in solving a
perplexing problem than in immortality. Possibly he took
it for granted that European scientists were already
three steps ahead of him.

By July he had prepared a manuscript describing all his

exciting experiments of the past two years, and
including specific instructions for setting up a lightning
rod on a tower or steeple, even to the necessary feature
of a grounding wire. “Let the experiment be made,” he
had said. He did not make it himself, not then. For one
thing, he was waiting for a spire to be erected on the
top of Christ Church, from which he wished to make his
first try of drawing lightning from the skies. Also, in
spite of his alleged retirement, his days were becoming
increasingly filled with public duties.

He still had the Gazette and Poor Richard’s Almanack to

publish and edit. Beginning in 1748, he served on the
City Council. Since 1749 he was Grand Master of the
Masons. In 1751 he was made an alderman and a
member of the Pennsylvania Assembly, where previously
he had served as clerk.

In 1750, an American Philosophical Society member, Dr.

Thomas Bond, came to him for help in starting a
hospital for the sick and the insane. Hitherto those who
could not pay for medical care had no choice but the
prison or the almshouse. The need was urgent but Dr.
Bond had failed to arouse interest in his project.

“Those whom I ask to subscribe,” he confided to 57

Franklin, “often ask me whether I have consulted you
and what you think of it. When I tell them I have not,
they don’t subscribe.”

Franklin knew promotion methods as Dr. Bond did not,

and began by calling a meeting of citizens. Under his
impetus the list of subscribers grew, though not until
May 1755 was the cornerstone of the Pennsylvania
Hospital laid on Eighth Street between Spruce and Pine.
Nearly thirty years later, when Dr. Benjamin Rush joined
the staff, the “lunatics” at Pennsylvania Hospital
received the first intelligent care available in America
and, with few exceptions, in the world.
Franklin was also busy during this period in the
formation of America’s first insurance company
(stemming from a meeting of Philadelphia businessmen
in 1752), and was taking the lead in organizing an
expedition in search of a Northwest Passage, under
Captain Charles Swaine, America’s first voyage of Arctic
exploration.

In the category of pleasure were the infrequent periods

he spent on his Burlington farm, where he raised corn,
red clover, herd grass and oats, recording with scientific
precision the effects of frost and the results obtained
from different types of soil. He was one of the earliest
Americans to think of agriculture as a science. He never
could persuade his farmer neighbors to follow his
example. They held that the ways of their forefathers
were inevitably the best.

It may have been at his farm that he made his

experiment on ants. Some ants had found their way into
an earthen pot of molasses. He shook out all but one
and hung the pot by a string to a nail in the ceiling.
When the ant had dined to its satisfaction, it climbed up
the string and down the wall to the floor. Half an hour
later, he noted a swarm of ants retracing its course back
to the pot—exactly as though their comrade had
verbally informed them where to go for a good meal.

There were few mysteries of nature on which at one 58

time or another Franklin did not direct his attention.
More often than not, he wrote his speculations in long
and entertaining and gracefully phrased letters to his
friends, men and women alike.

If he was not impatient to learn what Peter Collinson

thought of his proposed lightning rods, it was simply
that he had no time for impatience. The truth was that
Collinson had found his paper fascinating and had even
read it to the Royal Society. As the Society members
remained skeptical and unimpressed, in 1751 he
arranged for it to be printed in a pamphlet
—“Experiments and Observations on Electricity, Made at
Philadelphia, in America.” Dr. John Fothergill, a London
physician, wrote the preface. The pamphlet was
translated into French the next year, creating immediate
excitement.

Three French scientists, the naturalist Count Georges

Louis Buffon, Thomas François d’Alibard, and another
named de Lor, resolved to carry out the experiment on
drawing lightning from the skies, which Franklin had
outlined.

It was d’Alibard who succeeded first. At Marly, outside

of Paris, he set up a pointed iron rod forty feet long, not
on a church steeple as Franklin had recommended, but
simply on a square plank with legs made of three wine
bottles to insulate it from the ground. During a
thunderstorm, on May 10, 1752, a crash of thunder was
followed by a crackling sound—and sparks flew out from
the rod. Here then was absolute proof that Franklin was
right. Lightning and electricity were identical.

De Lor repeated the experiment in Paris eight days later. 59

Louis XV, King of France, was so moved that he sent
congratulations to the Royal Society, to be relayed to
Messieurs Franklin and Peter Collinson. The first
successful experiment in London was made by John
Canton. Soon it was being repeated throughout Europe.
The name of Benjamin Franklin was on everyone’s
tongue.
No news of all this had yet been brought on the slow
sailing ships when, in June 1752, Franklin decided not
to wait for the completion of the Christ Church spire for
his experiment. He had another scheme. Why not try to
draw electricity from the skies with a kite?

“Make a small cross of two light strips of cedar, the arms

so long as to reach to the four corners of a large thin
silk handkerchief when extended; tie the corners of the
handkerchief to the extremities of the cross.” Thus he
later described the body of this world famous kite. Like
ordinary kites, it had a tail, loop, and string. At the top
of the vertical cedar strip, he fastened a sharp pointed
wire about a foot long. At the end of the string he tied a
silk ribbon. He fastened a small key at the juncture of
silk and twine.

With this child’s plaything, he and his tall full-grown son,

William, took off across the fields one threatening
summer day. They let the wind raise the kite into the air
and they waited. Even before it began raining, Franklin
observed some loose threads from the hempen string
standing erect. He pressed his knuckle to the key—and
an electric spark shot out. There were more sparks
when the thunderstorm began. After the string was wet,
the “electric fire” was “copious.”

He must have grinned triumphantly at William, and

perhaps said casually, “Well, Billy, we’ve done it.”

There is no evidence that he realized his experiment 60

might be dangerous, even deadly.

The first account of the “Electrical Kite” appeared five

months later in the October 19, 1752, issue of the
Gazette. Poor Richard’s Almanack for 1753 contained
complete instructions on how to build a lightning rod.
He had already put one up on his own chimney. It had
small bells which chimed when clouds containing
electricity passed by. The bells rang in his house for
years.

News of his triumphs abroad were now flooding in. The

praise of the French king, he wrote a friend, made him
feel like the girl “who was observed to grow suddenly
proud, and none could guess the reason, till it came to
be known that she had got on a new pair of garters.”
The Royal Society, making up for lost time, published an
account of his kite in Transactions, their official paper,
and in November 1753, gave him the Copley gold medal
for “his curious experiments and observations on
electricity.” They conservatively held off making him a
member of the Society until May 29, 1756. At home,
Harvard, Yale, and William and Mary College in turn
gave him honorary degrees of master of arts.

While these and other tributes were being heaped on

him, he was launching into a new profession—that of
military expert and officer.

61
 6
A BRIEF MILITARY CAREER

In 1753, trouble was brewing once more between Great

Britain and France, with the colonists caught in the
middle. While English subjects in America were as yet
confined to a narrow strip along the Atlantic, France
held Canada and the St. Lawrence Valley to the north;
New Orleans and the great Louisiana territory in the
south. By right of early explorations, the French also
claimed the rich Ohio Valley region and were building
forts along the Ohio and Allegheny rivers. The British
considered these forts an intrusion on their territory.

As the situation grew more tense, both British and

French courted the favor of the Indians. In Pennsylvania
this would have been easier had the policy of William
Penn been followed; he had gone further than any other
white man in establishing friendly Indian relations.
Unfortunately, much of his work had been undone by
his son Thomas, in the episode known as the Walking
Purchase.

To make room for his immigrants, William Penn had 62

once purchased a tract of land from the Indians to
extend “as far as a man could walk in three days.” In
1683, he had leisurely walked out a day and a half of
this purchase, some twenty-five miles. In 1737, fifty
years later, Thomas Penn decided to take up the rest of
the Walking Purchase. He hired three athletes to do the
walking for him. In a day and a half, they managed to
cover eighty-six miles. The Indians had never forgiven
this underhanded trick.

It was partially to undo this bad feeling that in

September 1753 Franklin and several other
commissioners were sent by Governor James Hamilton
to Carlisle, some 125 miles west of Philadelphia, to meet
with chiefs of the Delaware and Shawnee Indians and
the Six Nations (the name given to the united Iroquois
tribes).

Franklin had never been so far inland before nor had he

any previous dealings with the original Americans. He
was impressed with the ceremonial exchange of gifts
and greetings which preceded the actual conference.
These “savages” of whom he had heard such
disparaging things had customs very different from
those of the white man, but “savage justice,” as he was
to write later, had as much to recommend it as “civilized
justice.”

The grievances presented by the chiefs after the

conference began he found reasonable. They wanted,
from the white man, fewer trading posts and more
honest traders. They wanted to be sold less rum, which
was ruinous to the braves, and more gunpowder, which
they needed for hunting. The commissioners promised
to do their best and, as they had been authorized to do,
offered the Indians protection from the French, in return
for their loyalty. Unfortunately, neither colonies nor
British were in a position to guarantee such protection.
Franklin returned from Carlisle to learn that he had been 63
appointed deputy postmaster, with William Hunter of
Williamsburg, of all the North American provinces. He
had the prestige of being an officer of the Crown though
the pay was nominal—only 600 pounds a year divided
between him and Hunter should the service make a
profit—and the work was considerable, for Hunter was
ill and could give little help.

He could and did provide his family with jobs. William,

his son, became postmaster of Philadelphia, Franklin’s
former job. William later turned this post over to a
relative of Debby’s who in due time was succeeded by
Franklin’s brother Peter. He appointed another brother,
John, postmaster of Boston. At John’s death his widow
succeeded him, thought to be the first American woman
to hold a public office.

Not only his family but all of America profited by

Franklin’s appointment. Horseback riders carried mail in
colonial America. Delivery was slow, irregular and costly.
Franklin acted as an efficiency expert. He increased mail
deliveries from Philadelphia to New York from once a
week to three times a week during the warmer six
months of the year and he made sure his riders did the
route twice a week in the winter except in the worst
weather. In time he visited all the post offices of the
colonies, studied their local problems, surveyed roads,
ferries, and fords. He started America’s first Dead Letter
Office, and gave patrons other services they had never
had before. By the time he had held the post eight
years, not only could he and Hunter collect their full
salaries but there was a surplus for the London office,
the first time it had ever profited from its American
branch.
Late in 1753, Governor Robert Dinwiddie of Virginia sent 64
young Major George Washington on a journey to the
French Fort Le Boeuf (now Erie, Pennsylvania) to order
the French to evacuate. They chose to ignore the
warning.

Franklin attended another conference with the Six

Nations, held at Albany, New York, in June 1754,
attended by commissioners from seven colonies. In
regard to Indian relations, the Albany conference was
no more successful than the one at Carlisle. Afterward
the Indians claimed they had been persuaded to deed a
tract of land whose boundaries they had not grasped
and that the deed was irregular since, contrary to the
Six Nations’ custom, it gave away land of tribes whose
representatives had not signed the deed.

Thus the two meetings had the opposite effect of what

had been hoped. They succeeded only in antagonizing
the Indians. Many of them decided to support the
French, as the lesser of the two white evils.

It is most unlikely that Franklin suspected any wrong

being perpetrated on the Indians. During the Albany
conference he presented to his fellow commissioners a
plan which had its inspiration from Six Nations. If the
Iroquois tribes could work together harmoniously, why
should the American colonies, allegedly civilized, always
be quarreling? Accordingly, he proposed they form a
confederacy under a single president-general appointed
by the Crown.

The commissioners approved wholeheartedly but that 65

was as far as he got. When his plan was presented to
the assemblies of the various colonies, it was rejected
as being too dictatorial. The Crown opposed it as being
too democratic. In a final effort to make his point he
published in the Gazette America’s first cartoon, a
drawing of a snake chopped in eight pieces, each
marked with the initials of different colonies. “Join or
Die” read the caption. But he was several years in
advance of the times.

Even while the Albany conference was under way, seven

hundred French soldiers and Indians forced the
surrender of Fort Necessity, a small barricade fifty miles
from Wills Creek, held by George Washington, now a
colonel, and a scant 400 men. The nine-year French and
Indian Wars were unofficially under way.

In December, six months later, General Edward

Braddock landed in Virginia with two regiments of
British regulars. They had come to take the French Fort
Duquesne, located on the forks of the Ohio (where
Pittsburgh now stands). The Pennsylvania Assembly
sent Franklin to meet the general at Frederickstown and
offer his services as postmaster. Franklin with his son
William spent several days with Braddock. He found the
general a master of European military strategy but more
than a little arrogant.

“After taking Fort Duquesne,” Braddock announced one

night at dinner, “I will proceed to Niagara; and, having
taken that, to Frontenac, if the season will allow time;
and I suppose it will, for Duquesne can hardly detain
me above three or four days.”

In his mind, Franklin pictured the long line of Braddock’s

army marching along a narrow road cut through thick
woods and bushes, and he was uneasy. He was sure, he
told the general, that there would be scant resistance at
Duquesne, if he arrived there. The danger would be
Indian ambush on the way.

Braddock smiled patronizingly. “These savages may, 66

indeed, be a formidable enemy to your raw American
militia, but upon the king’s regular and disciplined
troops, sir, it is impossible they should make any
impression.”

Franklin did not press his doubts. It would have been

improper for him to argue with a military man about his
own profession. Braddock was only too glad to let
Franklin hunt up some transport wagons for him. This
he did by distributing circulars through Lancaster, York
and Cumberland counties. Within two weeks
Pennsylvania farmers had come through with the loan of
150 wagons and 259 horses. Of the 1,000 pounds due
the owners in payment, Braddock paid 800 and Franklin
advanced the extra 200 pounds on his own. Since the
farmers knew and trusted him, he, rather than
Braddock, gave them his bond for the full cost.

After he returned to Philadelphia, he persuaded the

Assembly to donate twenty parcels for the regiment
officers, each containing six pounds of sugar, a pound of
tea, six pounds of coffee, six pounds of chocolate, as
well as biscuit, cheese, butter, wine, cured hams. He
sent along other supplies for the soldiers, advancing
1,000 pounds more of his own money to cover the
costs. Barely had he been reimbursed for his expenses
thus far, when the disastrous news broke.

Braddock’s army—some 1,400 British regulars and 700

colonial militiamen—was ambushed by a force of
French, Canadians, and Indians on July 9, 1755, when
they were within seven miles of Fort Duquesne. Terrified
at the shooting from this invisible enemy, the regulars
panicked. Nearly a thousand were killed or wounded,
including most of the officers. George Washington, who
was serving as Braddock’s aide, stayed to fight a valiant
rear guard action. Braddock was mortally wounded,
dying four days later.

At the start of the fray, the drivers took one horse from 67
each team and raced off, leaving wagons, food parcels
and provisions to the attackers. Since Franklin had given
bond, the wagon owners soon appeared, demanding
recompense for their losses—a total of some 20,000
pounds. He faced ruin until October when the new
British commander-in-chief, Governor Shirley, authorized
government payment of the debt.

In the midst of that summer’s harassment and disaster,

there was one pleasant interlude. On a trip to visit
Rhode Island post offices, Franklin met a delightful
young lady named Catherine Ray. Middle-aged and
tending to stoutness as he was, she lavished affection
on him, not as a suitor but as someone to whom she
could confide her innermost thoughts. Though he saw
Catherine only infrequently after that meeting, she later
married a worthy young man named William Greene by
whom she had six children—she and Franklin wrote
each other lengthy and intimate letters as long as they
lived. Until he met her, apart from Debby, his friendships
had all been with men. Beginning with Catherine, he
had many women friends, who found in him a rare
understanding of their qualities of mind and spirit.

The defeat of Braddock taught the colonists that the

British military was not as invincible as they had been
led to believe. Many more Indians joined the French,
deciding they were most likely to win. In the summer of
1755, Indian raiders were attacking isolated farms less
than 100 miles from Philadelphia. It was obvious that
once again Pennsylvania must provide its own defense.

A bill to vote 60,000 pounds for the militia was 68

presented to the Pennsylvania Assembly. At first the
Quakers opposed it, but with great tact Franklin won
from them a concession that even though they bore no
arms themselves they would not object if others did so.
There was still more dissension on the subject of taxes.
Franklin and many others believed that the taxes should
be raised from all the landholders in the province. The
lawyer for “the proprietors” claimed that the Penn family
should be exempt from such taxes, as they always had
been. He was supported by the conservatives in the
Assembly and by Governor Robert Hunter Morris, who
owed his appointment to the Penns.

Eventually the Penns compromised by offering 5,000

pounds toward the militia as a gift. The question as to
whether or not their vast lands should be taxed
remained unsettled, to trouble the future. Thomas Penn,
who was living in London, was duly informed that
Benjamin Franklin was a crafty man who could bend the
Assembly to his will.

On November 24, 1755, a Shawnee war party burned

down the Moravian village of Gnadenhuetten, 75 miles
from Philadelphia, killing all the inhabitants except a few
who escaped into the forests. The crime was the more
appalling since the Moravians were as opposed to
violence as the Quakers. They were a gentle, devout
people who had befriended the Indians. The next day
the Assembly appointed Franklin to head a committee of
seven to manage the funds for the defense. More
responsibilities on his shoulders, more decisions to
make, arguments to settle, hotheads to calm down.

“All the world claims the privilege of troubling my

Pappy,” wailed Deborah to a clerk named Daniel Fisher
whom Franklin had just hired.

A few weeks later Franklin set out on horseback with 50 69

cavalrymen to recruit volunteers, and check on defenses
in outlying districts—a strenuous assignment for a man
nearly fifty and sedentary in his habits. William served
as his aide. Theoretically, James Hamilton, a former
governor, was in charge, but after a few days he quietly
yielded the leadership to Franklin.

Their first stop was Bethlehem, the chief Moravian

settlement. Franklin had expected them to be as
opposed to military defense as the Quakers. On the
contrary, they were determined to avoid a tragedy such
as that at Gnadenhuetten, had built a stockade around
their principal buildings, brought in arms from New
York, and were even arming their women with small
paving stones to throw out the windows should any
marauding Indians approach.

“General Franklin,” the Moravians insisted on calling the

head of the Philadelphia expedition.

They rode on to Easton next, to find a town in a state of

panic and disorder with no discipline at all. Refugees
filled the houses. Food was almost gone. There was
drinking and rioting. Franklin organized a guard, put
sentries on the principal street, set up a patrol, had
bushes outside of town cleared away to avert their use
as ambush, and enlisted some two hundred men into
the provincial militia.
They visited other towns, arriving at the ruins of
Gnadenhuetten in the bitter cold of January. After the
mournful chore of burying the dead, the men set to
building a stockade—felling pines, placing them firmly in
the ground side by side. Franklin, with his passion for
collecting facts, noted that it took six men six minutes
to fell a pine of 14-inch diameter, and he observed that
his men were more cheerful on the days they worked
than when, because of rain or snow, they had to sit idle.

Supplies were running low when provisions arrived from 70

Philadelphia, including roast beef, veal, and apples from
Deborah. To reassure her, he wrote that he was sleeping
on a featherbed under warm blankets. The truth was
that, like his men, he slept on the floor of a hut with
only one thin blanket. The stockade, finished at last,
was 450 feet in circumference, 12 feet high, and had
two mounted swivel guns but no cannon.

They were aware of the danger lurking in the dense

forest. On a patrol, Franklin found the remains of Indian
watches. For their fires they dug holes about three feet
deep. The prints in weeds and grass showed they had
lain in a circle around the fire holes, letting their feet
hang over to keep warm. At a short distance, neither
flame, sparks, nor smoke could be seen. But the
Indians, not then nor later, risked an attack.

Franklin’s militia did no fighting but they turned

defenseless regions into defensive ones. They had built
two more stockades at Fort Norris and Fort Allen, when
Franklin was called back to Philadelphia early in
February for a special Assembly meeting. To have a
good bed again seemed so strange, he hardly slept all
night long.
On his return he was appointed a militia colonel.
Following his first review of his regiment, the men
accompanied him to his house and saluted him with
several rounds of fire, incidentally breaking some glass
tubes of his electrical apparatus. The following day
when he set off for Virginia on post office business, 20
officers and some 30 grenadiers escorted him to the
ferry, the grenadiers riding with drawn swords in a
ceremony reserved for persons of great distinction.
When Thomas Penn in England learned of this tribute,
he was furious. No grenadiers had ever drawn their
swords for him.

As for Governor Morris, he suavely suggested that 71

Franklin and his command should try to take Fort
Duquesne, which Braddock had failed to take, promising
him a general’s commission. Franklin firmly declined. He
had no illusions about his military ability and likely
suspected Morris of wishing to be rid of him. (Fort
Duquesne was eventually captured in 1758, in an
expedition led by British Brigadier General Forbes;
George Washington hoisted the British flag over the
fort’s ruins.)

In August 1756, following a declaration of war on the

Delaware, the new governor, William Denny, offered
large bounties for “the scalp of every male Indian
enemy above the age of twelve years,” and smaller
bounties for “female Indian prisoners and youths under
eight.” Franklin, like the majority of the Assembly
members, was outraged at this barbarity, and disgusted
with the conduct of the proprietors and their
representatives. Early in 1757, a vote was passed to
send Franklin to England, as official agent of
Pennsylvania, there to present to Parliament and the
King a petition of grievances against the Penns.
Debby would not go with him. She was frightened to
death of the sea. He did take William, who was radiant
at seeing England. By April they were in New York,
ready to catch their ship. Packets for England were in
charge of Lord Loudon, the new commander-in-chief, an
amiable person with all the time in the world to listen to
complaints, indulge in long conversations, and to write
endless notes. Not until he had finished this mysterious
correspondence, would he permit the fleet to depart.
For more than two months, Franklin and his son waited,
restless and impatient and helpless.

There was plenty of time to puzzle about the errors of 72

the British. Why they should send to the colonies an
arrogant man like General Braddock, a dawdler like
Loudon, governors like the dishonest Sir William Keith,
or Morris and Denny, who were far more interested in
protecting the rich proprietors than in the welfare of the
colonists. But then the reason for Franklin’s voyage was
to correct such mistakes. He had no doubt that the King
and the mighty Parliament would be glad to listen to
him.

73
 7
THE BATTLE WITH THE PENNS

During the voyage to England, Franklin wrote a preface

for his 1758 Almanack. In the form of a letter from
“Poor Richard” to his “Courteous Reader,” it told of a
sermon on frugality and industry, which Poor Richard
had heard in the market place by “a plain clean old man
with white locks” called Father Abraham. He was most
flattered to find that Father Abraham was quoting him,
Poor Richard, at every other breath.

As Poor Richard says: Many words won’t fill a

bushel.... God helps them that help themselves....
The sleeping fox catches no poultry.... Early to bed,
early to rise, makes a man healthy, wealthy, and
wise.... For want of a nail the shoe was lost; for
want of a shoe the horse was lost; and for want of
a horse the rider was lost....

As Poor Richard says: Many a little makes a

mickle.... Fools make feasts, and wise men eat
them.... Pride that dines on vanity, sups on
contempt.... ’Tis hard for an empty bag to stand
upright.... If you will not hear reason, she’ll surely
rap your knuckles...
All the nuggets of wise counsel which he had dropped in 74
his Almanack in the twenty-five years of its existence,
Franklin gathered for Father Abraham’s speech. Omitted
were the racy ballads, verses, broad humor and jokes
which had made the Almanack a potpourri where every
man could find something to his taste. Only at the end
was a touch of Franklin’s sly wit. Following Father
Abraham’s sermon, Poor Richard watched disconsolately
as the village folk dispersed to spend their hard-earned
money as foolishly as ever on the marketplace wares.
The only one to take the sermon to heart was Poor
Richard himself who had come to buy material for a
new coat but left, “resolved to wear my old one a little
longer.”

Father Abraham’s speech was later published under the

title of “The Way to Wealth.” It was reprinted in many
editions and translated in many languages, and it won
the author almost as much fame as his discoveries in
electricity.

Peter Collinson met Franklin and his son in London,

where they arrived on July 26, 1757, taking them to his
home. No doubt he and Franklin discussed electricity
until very late, with William only half listening and more
or less bored. The next day, a printer named William
Strahan, with whom Franklin had corresponded some
fourteen years but never met, called on him.

“I never saw a man who was, in every respect, so 75

perfectly agreeable to me,” Strahan wrote Deborah
Franklin of this meeting, adding that William was “one
of the prettiest young gentlemen I ever knew from
America.”
Deborah likely scowled. It was just like that artful lad to
ingratiate himself so quickly.

A few days later father and son rented four rooms from
a widow, Mrs. Margaret Stevenson, who lived with her
young daughter Polly in a substantial mansion on 7
Craven Street, Strand. This was to be Franklin’s English
home, which over the years became almost as dear to
him as his Philadelphia one.

He had brought two servants with him. One of them,

Peter, served him faithfully, though the other, a slave,
ran away shortly after their arrival. Franklin’s post as
Massachusetts agent required a bit of pomp. He wore a
wig in the latest fashion, silver shoe and knee buckles
and purchased linen for new shirts. Later he rented a
coach.

Barely was he settled when he was invited to visit Lord

George Grenville, president of the Privy Council and one
of England’s most important statesmen. This was
Franklin’s first test in holding his own with persons more
steeped than he in political intrigue.

Lord Grenville received him with great civility,

questioned him at length about American affairs, and
then announced that the colonists had some erroneous
notions he felt duty bound to correct:

“You Americans have wrong ideas of the nature of your

constitution; you contend that the King’s instructions to
his governors are not laws, and think yourselves at
liberty to regard or disregard them at your own
discretion. You must be made to understand that the
King’s instruction are The Law of the Land.”
This was simply not true. The King’s instructions were 76
laws in the colonies only if they received the approval of
the local Assembly. In the same way, laws passed by a
colonial Assembly had to be submitted to the King
before they became final. That was why Franklin was in
England, to get the King’s approval of the Assembly
decision on the Penns.

Sure as he was of his facts, he voiced his opinion in the

manner he had learned from Socrates: “It is my
understanding that ...”

“You are totally mistaken,” Lord Grenville stated

patronizingly, when he had finished.

It was Franklin’s first experience with the contemptuous

attitude which certain of the British took in regard to the
colonists. He would later observe that “every man in
England seems to jostle himself into the throne with the
King, and talks of our subjects in the colonies.”

Around the middle of August he called on the Penn

family, at their stately mansion in Spring Garden. It had
seemed courteous to meet with them personally before
approaching higher authority. William Penn’s son
Thomas was there and probably Richard Penn and his
son, John. They received him with glacial politeness,
listened haughtily as he told them the Assembly’s
grievances, and just as haughtily denied that the
grievances were in any way justified.

Franklin pointed out that the Assembly was asking no

more than what William Penn had promised citizens
under his 1701 Charter of Privileges.

“My father granted privileges he had no right to grant,

according to the Royal Charter,” Thomas Penn
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