My research group studies the role of cytoplasmic tyrosine kinases in hematopoietic cells. We are currently focusing on the Src-family kinases; these proteins are believed to play critical roles in transducing signals from cell surface receptors to intracellular targets. We have taken a genetic approach to study these kinases by generating knockout mice that lack the Src-family kinases Hck, Fgr, Lyn and Src. Single mutant mice can then be interbred to generate mice lacking two or three members of this gene family. Hematopoietic cells from the mutant mice are then studied to ask which signaling pathways are affected by the mutations. Initial studies have revealed that Hck/Fgr double mutant mice have a novel immunodeficiency to bacterial infection that may be due to impaired neutrophil function. The neutrophils from these mice fail to become activated following binding of surface adhesion receptors (integrins). Biochemical experiments have confirmed that integrin-dependent signaling in myelomonocytic cells from Hck/Fgr mice is impaired. This results in impaired cytoskeletal structure in cultured hematopoietic cells and reduced immune function in vivo. Lyn deficient animals manifest defects in B-lymphocytes as well as myeloid cells. B-cells from the Lyn knockouts fail to mature appropriately and are turned over very rapidly. Surprisingly,lyn-/- mutant B-cells are hyper-responsive to antigen-receptor signaling, suggesting that this kinase has a predominate role in down-regulation of signal transduction. Biochemical experiments have confirmed that lyn-/- B-cells show evidence of increased signaling through the Ras-MAPK pathway. Lyn mice also manifest defects in bone marrow hematopoiesis that may be secondary to loss of negative regulation of signal transduction. These results suggest that Hck and Fgr may function predominately in the integrin-signaling pathway, while the Lyn kinase may function to down-regulate signaling by acting on phosphatases. These possibilities are currently being pursued. Lyn mice have also been crossed with Hck/Fgr mutants to generate triple mutants animals which are being characterized. These animals will be especially useful since their myelomonocytic cells fail to express any detectable Src-family kinases, thus avoiding the problem of redundant function between these kinases.
In addition to continued evaluation of the above knockout mice, other long term projects in the laboratory may include generation of novel mutant animals with defects in other signaling molecules or receptors involved in hematopoietic cell development. In particular, we are interested in using site-specific recombination strategies to make animals with mutations restricted to specific hematopoietic lineages or that result in recombination between different gene- family members. We are also attempting to generate hematopoietic cell lines from the available mutant mice for further in vitro studies. In general, most projects related to blood cell development or immune function can be conducted in our laboratory.
Chan, V.W.F., Meng, F., Soriano, P., DeFranco, A.L., and Lowell, C.A. (1997).
Characterization of the B-Lymphocyte Populations in Lyn-Deficient Mice and the
Role of Lyn in Signal Initiation and Downregulation.
Submitted.
Meng, M. and Lowell, C.A. (1997). LPS-Induced Macrophage Activation and Signal Transduction in the Absence of Src-Family Kinases
Hck, Fgr and Lyn.
J.Exp.Med.,185, in press.
Lowell, C.A.,Fumagalli, L., and Berton, G. (1996).
Deficiency of Src-family Kinases p59/61hck and p58c-fgr Results in
Defective Adhesion-dependent Neutrophil Functions.
J.Cell Biology, 133, 895-910.