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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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