Role of Tyrosine Kinase in Infection Immunity

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To elucidate the function of tyrosine kinase Pyk2 in the activation of T cells, we established Jurkat transfectants overexpressing wild-type Pyk2, Pyk2 lacking 50 amino acids in its proline-rich domain, kinase-inactive Pyk2, and Pyk2 with amino acid substitution (^<402>Tyr-Phe or ^<881>Tyr-Phe).We investigated activities of MAP kinase, JNK and p38 kinase and IL-2 production after stimulation of these Jurkat transfectants by crosslinking of both T-cell antigen receptor and CD28.The results indicated that Pyk2 is involved in the IL-2 production through activation of JNK, and that both the kinase activity and the N-terminal tyrosine (^<402>Tyr) are indispensable for the production of IL-2.We found that Pyk2 is physically associated with Vav.As Vav is shown to have GEF activity for Rac and activation of Rac leads to activation of JNK, this finding may explain how JNK is activated by Pyk2.We further established WEHI231 (immature B cell) transfectants overexpressing either wild-type or kinase-inactive Pyk2, and analyzed the activation of MAP kinase, JNK and p38 kinase in these transfectants upon simulation with 1 mM H_20_2.The results demonstrated that Pyk2 is also involved in the oxigen stress-induced activation of JNK but not in the induction of apoptosis.We established experimental conditions for infection of MAIDS virus or Salmonella in Fyn- or Chk-knock out mice, respectively, and now are investigating abnormalities in immune system of these mutant mice after the infection.The chemokine receptor CXCR4 is expressed on the immature thymocytes.We found that Pyk2 is activated in Jurkat T cells after stimulation of CXCR4 with its ligand SDF-1.To clarify the role of Pyk2 and CXCR4, we are preparing transgenic mice which overexpress the dominant negative form of Pyk2 specifically in T cells by using a lck promotor-containing expression vector.