Many important processes are regulated by protein-tyrosy phosphorylation, which is regulated by protein-tyrosyl kinases (PTKs) and protein-tyrosyl phosphatases (PTPs). In hematopoiesis, homeostasis is maintained by the actions of growth factors and cytokines, which signal through receptor PTKs or receptor-associated PTKs. For example, colony stimulating f actor 1 (CSF1), the major factor controlling the proliferation and differentiation of macrophages in cell culture and in vivo, transmits signals through the CSFIR, a receptor PTK. Abnormal PTKs can cause hematopoietic failure or, conversely, diseases of hyper-proliferation, such as myeloproliferative syndromes or leukemia. Chronic myelogenous leukemia (CML) results from a translocation that fuses BCR with C-ABL, resulting in a fusion protein, BCR-ABL, with dysregulated PTK activity. Although much as been learned about how PTKs regulate normal hematopoiesis, and how abnormal PTKs cause disease, little is known about the roles of specific PTPs in normal or patho-physiology. The goal of this research is to define the function of two PTPs, SHPTP1 and SHPTP2, in CSF1R signaling and in BCR-ABL transformation. Genetic and biochemical data suggest SHPTP1 is a negative regulator of many PTK pathways, whereas SHPTP2 may play a positive role. We have found that macrophages from motheaten (melme) mice, which lack SHPTP1, hyper-proliferate in response to CSF1. SHPTP1, hyper-proliferate in response to CSF1. SHPTP1 appears to negatively regulate CSF1R signaling by a novel mechanism involving recruitment of substrates to the SHPTP1 catalytic domain via GRB2. We have also found that SHPTP1 and SHPTP2 bind BCR-ABL, although they likely have distinct biochemical and biological consequences. SHPTP2 also binds to a 97kD protein (P97) in BCR-ABL transformed cells which may be identical to a protein that binds SHPTP2 upon cytokine stimulation. We will define the molecular details of SHPTP1 regulation of the CSFIR. The site(s) of SHPTP1 tyrosyl phosphorylation in response to CSF1 will be mapped and mutated to prevent tyrosyl phosphorylation and/or GRB2 binding. Thr properties of these mutants will be assessed using a novel transient reconstitution assay in me/me macrophages. The 130 kD pY-protein that binds to the SH2 domains of SHPTP1 will be identified and its function determined. Whether all downstream CSFIR pathways are equally affected by loss of SHPTP1 will be ascertained, as will the reason(s) for the shortened doubling time in me/me macrophages. The importance of the CSFIR pathway to the overall me/me phenotype will be assessed by a transgenic reconstitution strategy and by genetic analysis. The binding sites for both SHPTPs on BCR-ABL will be mapped and the domain(s) of SHPTP2 required to bind p97 will be elucidated. The biological properties of BCR0ABL mutants unable to bind SHPTP1 or SHPTP2 mutants unable to bind P97, will be tested in standard transformation assays and the effects of restoring SHPTP-1 expression to k562 cells, which lack SHPTP1 expression, will be determined. Finally, we will ask whether changes in SHPTP/BCR-ABL associations correlate with CML progression by assaying the status of these interactions in blasts and neutrophils from chronic and acute phase patients. These studies should yield new insights into how PTPs contribute to the control of normal hematopoiesis and its disruption in human disease.
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