Role of Tyrosine Kinase in Infection Immunity

为了阐明酪氨酸激酶Pyk2在T细胞活化中的功能，我们建立了过表达野生型Pyk2的Jurkat转染物，Pyk2在其富含脯氨酸的区域缺乏50个氨基酸，激酶无活性的Pyk2，以及氨基酸取代的Pyk2 （^<402> tyrr - phe或^<881> tyrr - phe）。我们通过t细胞抗原受体和CD28的交联，研究了这些Jurkat转染物刺激后MAP激酶、JNK和p38激酶的活性以及IL-2的产生。结果表明，Pyk2通过激活JNK参与IL-2的产生，并且激酶活性和n端酪氨酸（^<402>Tyr）对于IL-2的产生都是必不可少的。我们发现Pyk2在物理上与Vav相关。由于Vav对Rac具有GEF活性，Rac的激活导致JNK的激活，这一发现可能解释了JNK是如何被Pyk2激活的。我们进一步建立了过表达野生型或激酶失活型Pyk2的WEHI231（未成熟B细胞）转染物，并在1 mM H_20_2模拟中分析了这些转染物中MAP激酶、JNK激酶和p38激酶的激活情况。结果表明，Pyk2也参与氧胁迫诱导JNK的激活，但不参与细胞凋亡的诱导。我们分别在Fyn-敲除小鼠和chk -敲除小鼠身上建立了感染MAIDS病毒和沙门氏菌的实验条件，目前正在研究这些突变小鼠感染后免疫系统的异常情况。趋化因子受体CXCR4在未成熟胸腺细胞上表达。我们发现Pyk2在CXCR4及其配体SDF-1刺激后在Jurkat T细胞中被激活。为了明确Pyk2和CXCR4的作用，我们正在制备转基因小鼠，通过使用含有缺乏启动子的表达载体，在T细胞中特异性地过表达Pyk2的显性阴性形式。

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.