Translation initiation has been demonstrated to be a key step in cell proliferation, differentiation and apoptosis. However, the mechanism(s) by which translation initiation is regulated remains largely unknown. We have demonstrated that the translational repressor 4E-BP1 (eIF4E-binding protein 1) is dephosphorylated and therefore activated in response to the immunosuppressant drug, rapamycin, and that 4E-BP2 (eIF4E-binding protein 2) is regulated during T cell maturation. We also demonstrated a differential regulation of 4E-BP1 and 4E-BP2 during myeloid differentiation, with an activation of 4E-BP1 specific to the monocytic/macrophage differentiation pathway and an upregulation of 4E-BP2 upon granulocytic differentiation. This proposal is intended to develop a better understanding of the process of translation initiation, focusing specifically on the role of 4E-BP proteins, in T lymphocytes and monocytes. The project takes advantage of mouse models with targeted disruption of either 4E-BP1 or 4E-BP2 and of technologies for transcriptomic and proteomic analysis.
The specific aims are: (i) to identify genes that are differentially translated in response to rapamycin. We reported that rapamycin specifically inhibits eIF4E-dependent translation. In contrast, rapamycin increases internal initiation of translation, a mechanism independent of e1F4E activity and reported so far for only a few cellular mRNAs. Genes affected by rapamycin through translational control mechanisms, will be identified using an oligonucleotide array based approach for measuring total RNA and polysome-bound RNA levels and a proteomic approach for measuring changes in protein levels in response to rapamycin; (ii) to determine the role of 4E-BP1 in rapamycin signaling. For this purpose, we propose to investigate the effect of targeted disruption of the 4E-BP1 gene in a mouse model, on rapamycin signaling; (iii) to determine the role of 4E-BP2 in T cell maturation and apoptosis. We have reported that protein synthesis rates, eIF4E phosphorylation and 4E-BP2 expression are differentially regulated upon activation of human immature and mature thymocytes. We will investigate the effect of knocking-out 4E-BP2 gene in a mouse model, on T cell maturation and on their sensitivity to apoptosis; (iv) to determine the role of 4E-BP1 and 4E-BP2 in myeloid differentiation. We also reported specific regulation of 4E-BP1 and 4E-BP2 expression, during monocytic/macrophage or granulocytic differentiation pathways. We will identify genes whose translation may be regulated specifically or predominantly by 4E-BP1 or by 4E-BP2 in myeloid cells, using both oligonucleotide array based and proteomic approaches. These studies will provide novel and important insights into the contribution of 4E-BP1 and 4E-BP2 genes, to the changes in T cell and monocyte gene expression associated with their maturation, proliferation or apoptosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI050896-05
Application #
6845678
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Macchiarini, Francesca
Project Start
2002-02-01
Project End
2007-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
5
Fiscal Year
2005
Total Cost
$346,000
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Olson, Katie E; Booth, Garrett C; Poulin, Francis et al. (2009) Impaired myelopoiesis in mice lacking the repressors of translation initiation, 4E-BP1 and 4E-BP2. Immunology 128:e376-84
Grolleau, Annabelle; Bowman, Jessica; Pradet-Balade, Berengere et al. (2002) Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics. J Biol Chem 277:22175-84