Normal hematopoiesis requires an intricate balance of cell proliferation, differentiation, and death. These processes are regulated by multiple signaling pathways that initiate with a protein-tyrosine kinase (PTK). Classical growth factor receptors are intrinsic PTKs, whereas cytokine receptors activate non-covalently associated PTKs. When these tyrosyl phosphorylation pathways are de-regulated, hematopoietic disease can result. For example, a number of different chromosomal translocations cause rearrangements in specific PTKs, which lead to their activation. These activated PTK fusions, which include Bcr-Abl, Tel-Jak and Tel-PDGFR cause myeloid leukemia. Leukemogenic retroviruses also can perturb tyrosyl phosphorylation. For example, the gp55 protein of Spleen Focus Forming Virus (SFFV) binds to and constitutively activates the erythropoietin receptor, leading to erythroid progenitor proliferation and ultimately, erythroleukemia. Protein-tyrosine phosphatases (PTPs) also regulate tyrosyl phosphorylation, but less is known about their biological functions. The long range goal of our research is to define the function of the SH2 domain-containing PTP SHP2 and its binding protein, Gab2, in normal hematopoiesis and disease. During this funding period, we purified and cloned Gab2, showing it is a scaffolding adapter related to Dos and Gab1. Gab2 is tyrosyl phosphorylated in response to multiple hematopoietic stimuli, and binds SHP2, PI3K and other signaling molecules. Acting though SHP2, Gab2 controls a novel pathway to immediate-early gene activation, whereas by binding to PI3K, it regulates the Akt pathway, particularly downstream of receptors that lack PI3K binding sites. Gab2 and SHP2 are activated constitutively in cells transformed by Bcr-Abl, Tel-Jak, Tel-PDGFR and SFFV, suggesting that they may play a role in leukemogenesis. In this continuation application, we will further analyze Gab2 and SHP2 in normal and abnormal hematopoiesis. We will use Gab2-/- mice to clarify why Gab2 is essential for eosinophil generation and for stress hematopoiesis. We will use inducible (floxed) SHP2 mice to circumvent the early lethality of SHP2 knockout mice and define the role of SHP2 in myelo- and erythropoiesis. We will further clarify the mechanism by which Gab2 and SHP2 are recruited to Bcr-Abl via Y177, a key residue in Bcr-Abl required for its full leukemogenic potential. Finally, using these novel reagents, and retroviral transduction/BMT assays we will determine the effect of Gab2 or SHP2 deletion on leukemogenesis by Bcr-Abl, Tel-PTK fusions, and SFFV. Our results should yield new insight into how specific PTK/PTP interactions regulate normal hematopoiesis and how these interactions are involved in stress hematopoiesis and leukemia.
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