Table Of ContentProgress in Molecular and Subcellular Biology
Se ries Editors: W.E.G. Müller (Managing Editor), Ph. Jeanteur, 27
I. Kostovic, Y. Kuchino, A. Macieira-Coelho, R. E. Rhoads
Springer-Verlag Berlin Heidelberg GmbH
Robert E. Rhoads (Ed.)
Signaling Pathways
for Translation
Stress, Calcium, and Rapamycin
With 27 Figures
Springer
Professor Dr. ROBERT E. RHOADS
Department of Biochemistry and Molecular Biology
Louisiana State University Health Sciences Center
Shreveport, LA 71130
USA
ISSN 0079-6484
Library of Congress Cataloging-in-PubIication Data
Signaling pathways for translation: stress, calcium, and rapamycin 1 Robert E. Rhoads (ed.).
p. cm. - (Progress in molecular and subcellular biology; 27)
Includes bibIiographical references and index.
1. Proteins - Synthesis. 2. Proteins - MetaboIism - Regulation. 3. Cellular signal
transduction. 4. Genetic translation. 5. Apoptosis. 6. Calcium - Physiological effect. 7.
Stress (Physiology) I. Rhoads, Robert E., 1944- Ir. Series.
QP551 .S558 2001
572'.645 - dc21
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ISBN 978-3-642-07505-6 ISBN 978-3-662-09889-9 (eBook)
DOI 10.1007/978-3-662-09889-9
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Preface
A diversity of stressful conditions cause rapid and severe inhibition of protein
synthesis in eukaryotic ceIls, in some cases resulting in cell death by apopto
sis (programmed cell death). Apoptosis has come to be recognised as an impor
tant physiological process in the regulation of growth, development and
differentiation. Until recently, relatively little attention had been paid to the
changes in protein synthesis during the various phases of apoptosis. The arti
des in the present volume are by major contributors to our understanding of
signaling pathways that result in the inhibition of protein synthesis. These
authors trace the downstream consequences of such stress conditions as virus
infection, heat shock, nutrient starvation, release of intracellular calcium ions,
and treatment with the immunosuppressant rapamycin.
Understanding the mechanisms by which the biosynthesis of pro teins is
regulated is important for several reasons. Pro tein synthesis consumes a major
portion of the cellular ATP that is generated. Therefore, small changes in
pro tein synthesis can have great consequences for cellular energy metabolism.
Translation is also a major site for control of gene expression, since messenger
RNAs differ widely in translational efficiency, and changes to the protein syn
thesis machinery can differentially affect recruitment of individual mRNAs.
From a medical standpoint, there is a dose but as yet incompletely understood
relationship between cell growth and protein synthesis. Understanding the
mechanisms regulating pro tein synthesis therefore holds promise for treat
ment of diseases that are characterized by uncontrolled cell proliferation as
weIl as those involving tissue degeneration.
The field of cellular signal transduction may be traced back as early as Earl
Sutherland's seminal discovery of 3', 5'-cydic AMP as a "second messenger",
but it has rapidly expanded over the past two decades. The area now indudes
cell surface receptors and a host of intermediate components that operate in
branching, and often interconnecting, pathways to trans mit an extracellular
signal to the ultimate cellular targets. Thus, an encoded chemical message is
translated into the language of the cell by altering a biochemical activity. The
complexity of the networks thus far elucidated permits multiple simultaneous
signals to be integrated and also allows a single signal to modulate a variety of
target activities. Differences in signaling pathways in various cell types allows
for ceIl-specific responses to the same agonist.
The process of protein synthesis is itself one of the most complex bio
chemical achievements of living ceIls, resulting in the translation of an encoded
VI Preface
genetic message into proteins that carry out both structural and functional
roles. This rapid and highly accurate process requires the coordinated action
of roughly 150 different polypeptides and 70 different RNAs. Discovering how
extracellular signals might regulate such an intricate and complex process rep
resents an enormous challenge.
Despite such complexity, considerable progress has been made regarding
this important question. Surprisingly, only a few of the many components of
the pro tein synthesis machinery have been implicated as targets of signaling
pathways. The majority of these involve individual polypeptide components of
the initiation and elongation factors. The change in activity of the factor is gen
erally accompanied by phosphorylation of the factor or phosphorylation of a
bin ding partner, changing either the intrinsic activity of the factor or the affin
ity of its association with the binding partner. In several cases, the end result
of a signaling cascade is proteolysis of the factor.
Given the subtlety and complexity of both the network of signaling path
ways and the machinery for protein synthesis, it is likely that our current
knowledge represents merely"the tip of the iceberg". It is the authors des ire
that the current work will stimulate further research in this interesting and
important field.
Shreveport, Louisiana, USA ROBERT E. RHOADS
May 2001
Contents
The Regulation of eIF4F During Cell Growth and Cell Death
Simon J. Modey
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . 1
2 Mechanism and Regulation of Initiation
of Protein Synthesis .................................. . 1
2.1 The mRNA-Binding Initiation Factors ................... . 3
2.1.1 Initiation Factor eIF4E ................................ . 3
2.1.2 eIF4E Phosphorylation ................................ . 5
2.2 Initiation Factor eIF4A ................................ . 5
2.3 eIF4B and eIF4H .................................... . 6
2.4 Scaffold Protein eIF4G ................................ . 7
2.5 Poly(A)-Binding Protein (PABP) ........................ . 7
2.6 eIF4E Binding Proteins ............................... . 8
3 The Regulation of eIF4F Complex Levels ................. . 9
3.1 eIF4F: An Overview .................................. . 9
3.2 eIF4F and Increased Rates of Protein Synthesis ............ . 10
3.3 eIF4F and Decreased Rates of Protein Synthesis ............ . 12
3.4 Internal Ribosome Binding, eIF4E, eIF4G Integrity
and Reduced Levels of eIF4F ........................... . 14
3.4.1 Initiation on Viral Internal Ribosome Entry Sites ........... . 14
3.4.2 Potential IRES in Capped, Cellular mRNAs ................ . 14
4 Cell Death and the eIF4F Complex ...................... . 17
4.1 Apoptosis: An Overview ............................... . 17
4.1.1 Apoptosis and Protein Synthesis Rates ................... . 18
4.2 Apoptosis and the Initiation Factors Involved in Binding
mRNA to the Ribosome ............................... . 18
4.2.1 eIF4GI and eIF4GII .................................. . 18
4.2.2 eIF4B, eIF3p35, 4E-BPI and eIF2a ....................... . 20
4.3 Possible Mechanisms of Translational Control
During Apoptosis .................................... . 22
4.3.1 Cleavage of eIF2a, eIF4B, eIF3p35 and the Increased
Binding of 4E-BPI to eIF4E ............................ . 23
4.3.2 Cleavage of eIF4G .................................... . 24
References .......................................... . 26
VIII Contents
Regulation of the Activity of Eukaryotic Initiation Factors
in Stressed Cells
Gert C. Scheper, Roel Van Wijk, and Adri A.M. Thomas
1 Stress and Pro tein Synthesis ........................... . 39
1.1 Inhibition of General Pro tein Synthesis .................. . 39
1.2 Phosphorylation of eIF2 ............................... . 39
1.3 Inactivation of eIF2B ................................. . 41
1.4 Inactivation of the Cap-Binding Complex eIF4F ............ . 43
1.5 Dephosphorylation of eIF4E ........................... . 44
1.6 Binding of eIF4E to 4E-BPs ............................ . 44
2 Preferential Translation of HSP mRNAs .................. . 45
3 Experimental Procedures .............................. . 50
References .......................................... . 52
Initiation Factor eIF2a Phosphorylation in Stress Responses
and Apoptosis
Michael J. Clemens
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2 EIF2a Kinases and Their Regulation . . . . . . . . . . . . . . . . . . . . . . 60
2.1 Double-Stranded RNA-Activated Protein Kinase (PKR) . . . . . . . 61
2.2 The Saccharomyces cerevisiae Protein Kinase GCN2 . . . . . . . . . . 64
2.3 PKR-Like Endoplasmic Reticulum Protein Kinase (PERK) . . . . . 65
2.4 Haemin-Regulated Inhibitor (HRI) ....................... 66
3 Physiological Regulation of eIF2a Phosphorylation . . . . . . . . . . 67
3.1 Stress Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.1.1 Heat Shock .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.1.2 Nutrient Supply ...................................... 70
3.1.3 Regulation by Calcium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.1.4 The Relative Importance of eIF2a Phosphorylation
and Other Mechanisms in Stress Responses. . . . . . . . . . . . . . . . 71
3.2 Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4 What Are the Consequences of eIF2a Phosphorylation? ...... 76
5 Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Elongation Factor-2 Phosphorylation and the Regulation
of Protein Synthesis by Calcium
Angus C. Nairn, Masayuki Matsushita, Kent Nastiuk, Atsuko Horiuchi,
Ken-Ichi Mitsui, Yoshio Shimizu, and H. Clive Palfrey
Introduction ........................................ . 91
2 Phosphorylation of eEF2 and Regulation of Polypeptide
Elongation in Vitro ................................... . 92
3 Structure and Regulation of EF2 Kinase .................. . 95
Contents IX
4 Phosphorylation of EF2 Kinase by PKA and Other Kinases . . . . 102
5 Regulation of EF2 Kinase Turnover
bya Ubiquitination/Proteosome-Dependent Pathway ........ 103
6 Hormonal Control of eEF2 Expression and Phosphorylation . . . 105
6.1 Regulation of eEF2 Protein ............................. 106
6.2 Regulation of eEF2 Phosphorylation . . . . . . . . . . . . . . . . . . . . . . 107
6.3 Regulation of eEF2 Dephosphorylation . . . . . . . . . . . . . . . . . . . . 108
7 Relationship of eEF2 Phosphorylation to Protein Synthesis
and Growth in Proliferating Cells ........................ 109
8 Cell Cycle-Dependent Phosphorylation of eEF2 ............. 111
9 Phosphorylation of eEF2 in Neurons. . . . . . . . . . . . . . . . . . . . . . 114
10 eEF2 Phosphorylation and the Regulation of Local Pro tein
Synthesis in Neurons .................................. 116
11 eEF2 Phosphorylation and Glutamate-Mediated Control
of Protein Synthesis at Developing Synapses ............... 118
12 Concluding Remarks Concerning the Physiological Role
of eEF2 Phosphorylation ............................... 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Phosphorylation of Mammalian eIF4E by Mnk1 and Mnk2:
Tantalizing Prospects for a Role in Translation
Malathy Mahalingam and Jonathan A. Co oper
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
2 Mnks: Discovery and Kinase Activation ................... 132
3 Mnk1 Binding Proteins ................................ 134
4 Association With and Phosphorylation of the eIF4G Scaffold .. 135
5 The eIF4E Kinase? .................................... 136
6 Possible Consequences of eIF4E Phosphorylation ........... 137
7 Conclusion .......................................... 138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Control of Translation by the Target of Rapamycin Proteins
Anne-Claude Gingras, Brian Raught, and Nahum Sonenberg
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
2 Rapamycin (Also Known As Sirolimus or Rapamune) ........ 144
2.1 Discovery and Activity of Rapamycin .... . . . . . . . . . . . . . . . . . 144
2.2 Rapamycin-Related Compound FK506 .................... 145
3 Immunophilins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
3.1 FK506-Binding Proteins (FKBPs) . . . . . . . . . . . . . . . . . . . . . . . . . 146
3.2 Cyclophilins and Parvulins ............................. 147
4 Identification of Rapamycin Targets ...................... 147
4.1 Cloning ofYeast Target of Rapamycin (TOR) Proteins . . . . . . . . 147
4.2 Cloning of the Mammalian Target of Rapamycin Proteins . . . . . 149
5 Modular Structure of the TOR Proteins ................... 150
