SRC FAMILY PROTEIN TYROSINE KINASES IN HEMATOPOIESIS

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Cytoplasmic tyrosine kinases play critical roles in intracellular signaling in hematopoietic cells. To study the functions of these kinases in blood cells we have used gene targeting in embryonic stem cells to generate mice with mutations in several sec family genes. We have concentrated on the hck, fgr, lyn, and src genes, the kinases encoded by these genes have been implicated in signaling pathways elicited by cytokines, lipopolysaccharide, and crosslinking of IgM and Fc receptors. Analysis of monocytes and macrophages from hck-/- mice has revealed a reduced ability to phagocytose latex heads. However, no other myeloid cell phenotypes have been found suggesting that the deficiency of Hck is complemented by other Src family kinases. Interbreeding of single mutants to generate double mutant animals has confirmed that these kinases are, in part, functionally redundant. For example, hck-/--src-/- double mutants develop severe extramedullary hematopoiesis as a result of poor osteoclast function and osteopetrosis. Research in this project will focus on a continued characterization of the available single and double mutant animals. We hypothesize that the defects seen in hck-/-macrophages and hck-/-src-/- osteoclasts may result from impairments in cell adhesion and integrin receptor signaling. To test this, and to facilitate analysis of signal transduction in mutant cells, hematopoietic cell lines will be derived forma the mutant mice. To study the role of the Lyn kinase in B-cells and myeloid cells, lyn-/- mice are currently being developed. We hypothesize that a deficiency in Lyn will result in defective signaling though surface mu=chains producing in a block in B-cell development and/or function. Additionally, we will cross the lyn=/= mice to the hck=/= and src-/- mutants to look for novel phenotypes in myeloid cells. In order to study the role of Src-family kinases in megakaryocytopoiesis, we propose to generate mice lacking the Matk/Hyl kinase, which serves as a negative regulator of Src-family members in megakaryocytes and platelets. We postulate that such animals will manifest dysregulated signaling causing blocks in megakaryocyte development, platelet formation or platelet function. The goal of these studies is to use genetics in order to define the signaling pathways in which these kinases play physiologically significant functions. Ultimately, this approach will lead to a molecular understanding of the roles of tyrosine kinases in the regulation of hematopoietic cell growth, differentiation and function. These studies will be carried out in collaboration with Drs. DeFranco and Leavitt within the SCOR in Transfusion medicine.