The goal of this study is to identify the early signal transduction mechanisms by which GM-CSF stimulates the differentiation of hematopoietic cells. These complex signals appear to be generated by the interaction of specific domains of the alpha and beta chains of the GM-CSF receptor (R) and associated protein kinases. The analysis of GM-CSF-induced differentiation will be made possible by a unique cell line FDCP-1/19WT that grows in IL-3, but differentiates in GM-CSF. This cell line will be used to (1) map the specific domains of the alpha and beta chains which regulate differentiation, (2) determine the role of specific phosphorylation sites in the beta chain in the differentiation process, and (3) examine the need for alphaPK and JAK2 protein kinases, as well as the Ras pathway in the differentiation. The preliminary results and unique reagents generated in our laboratory will make this analysis possible. We have found that (1) the sort 54 amino acid intracytoplasmic tail of the alpha chain of the GM-CSFR is necessary for GM-CSF mediated differentiation, (2) a newly cloned protein kinase, alphaPK, binds to this alpha intracytoplasmic domain, and (3) a CD16/JAK2 fusion protein encoding only the kinase domain of JAK2 is sufficient to stimulate cell growth of GM-CSF-dependent cell lines. Using these reagents and the FDCP-1 19WT cells, we will generate a unique understanding of how GM-CSF signals hematopoietic cells to differentiate.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044741-06
Application #
2608445
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1992-03-01
Project End
2000-11-30
Budget Start
1997-12-01
Budget End
1998-11-30
Support Year
6
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Lilly, M; Sandholm, J; Cooper, J J et al. (1999) The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene 18:4022-31
Biggs, J R; Kraft, A S (1999) The role of the Smad3 protein in phorbol ester-induced promoter expression. J Biol Chem 274:36987-94
Matsuguchi, T; Lilly, M B; Kraft, A S (1998) Cytoplasmic domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor beta chain (hbetac) responsible for human GM-CSF-induced myeloid cell differentiation. J Biol Chem 273:19411-8
Matsuguchi, T; Kraft, A S (1998) Regulation of myeloid cell growth by distinct effectors of Ras. Oncogene 17:2701-9
Franklin, C C; Srikanth, S; Kraft, A S (1998) Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis. Proc Natl Acad Sci U S A 95:3014-9
Biggs, J R; Ahn, N G; Kraft, A S (1998) Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein. Cell Growth Differ 9:667-76
Matsuguchi, T; Zhao, Y; Lilly, M B et al. (1997) The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation. J Biol Chem 272:17450-9
Zhao, Y; Loyer, P; Li, H et al. (1997) Cloning and chromosomal location of a novel member of the myotonic dystrophy family of protein kinases. J Biol Chem 272:10013-20
Sakai, I; Kraft, A S (1997) The kinase domain of Jak2 mediates induction of bcl-2 and delays cell death in hematopoietic cells. J Biol Chem 272:12350-8

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