Role of Tyrosine Kinase in Infection Immunity

酪氨酸激酶在感染免疫中的作用

基本信息

批准号：
09836007
负责人：
MORIKAWA Yuko
金额：
$ 1.92万
依托单位：
The Kitasato Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09836007/
关键词：
Tyrosine Kinase Pyk2 Isoform Infection Immunity Salmonella AIDS Fyn Chk マクロファージ分化

项目摘要

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.

为了阐明酪氨酸激酶 Pyk2 在 T 细胞激活中的功能，我们建立了过表达野生型 Pyk2、富含脯氨酸结构域中缺少 50 个氨基酸的 Pyk2、激酶失活的 Pyk2 和具有氨基酸取代的 Pyk2 的 Jurkat 转染子 (^ <402>Tyr-Phe 或 ^<881>Tyr-Phe)。我们研究了 MAP 激酶、JNK 和 p38 激酶的活性通过 T 细胞抗原受体和 CD28 交联刺激这些 Jurkat 转染子后产生 IL-2。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 -末端酪氨酸(^<402>Tyr)对于IL-2的产生是不可缺少的。我们发现Pyk2与Vav有物理相关性。Vav被证明对Rac具有GEF活性并激活Rac导致JNK的激活，这一发现可能解释了Pyk2如何激活JNK。我们进一步建立了过表达野生型或激酶失活Pyk2的WEHI231（未成熟B细胞）转染子，并分析了MAP激酶、JNK和p38激酶的激活在这些转染子中，用 1 mM H_20_2 进行模拟。结果表明，Pyk2 也参与了氧应激诱导的 JNK 激活，但不参与诱导我们分别建立了Fyn或Chk敲除小鼠感染MAIDS病毒或沙门氏菌的实验条件，现在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表达在未成熟胸腺细胞。我们发现Pyk2在Jurkat T细胞中用其配体SDF-1刺激CXCR4后被激活。为了阐明Pyk2和CXCR4的作用，我们正在准备通过使用含有 lck 启动子的表达载体，转基因小鼠特异地在 T 细胞中过度表达 Pyk2 的显性失活形式。