Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion.
Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion.
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富含脯氨酸的酪氨酸激酶 2 调节 β2-整合素粘附后嗜酸性粒细胞的扩散和迁移。
DOI：
10.1165/rcmb.2008-0047oc
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发表时间：
2008
影响因子：
6.4
通讯作者：
Leff,AlanR
中科院分区：
文献类型：
--
作者：
Zhu,Xiangdong;Boetticher,Evan;Wang,Lin;Duan,Yingli;Learoyd,Jonathan;Leff,AlanR
我们研究了富含脯氨酸的酪氨酸激酶(PYK)2在β2整合素结扎后人血嗜酸粒细胞扩散和迁移中的作用。Western印迹分析表明，经IL-5、血小板激活因子(PAF)、甲酰-甲基亮氨酸-苯丙氨酸(FMLP)或Mn2+激活后，PYK2在Y402处被磷酸化激活。为了确定PYK2在调节嗜酸性粒细胞迁移中的作用，我们使用了一种可转导的显性负性PYK2抑制物，即TAT介导的显性负性C端PYK2的蛋白转导(TAT-PYK2-CT)，TAT-PYK2-CT是一种融合蛋白，其中TAT肽与C端PYK2融合。TAT-PYK2-CT可阻断β-2-整合素黏附引起的PYK2酪氨酸磷酸化，但不能阻断嗜酸性粒细胞与平板牛血清白蛋白的黏附。TAT-Pyk2-CT还可阻断IL-5、PAF或fMLP引起的嗜酸性粒细胞随后的扩散和迁移。FITC结合的鬼臼蛋白染色的弥漫性嗜酸性粒细胞延长并形成多个足底和片状脂膜，而非播散性嗜酸性粒细胞较小和圆形。TAT-PYK2-CT处理嗜酸性粒细胞对初始细胞极化无影响，但可阻断贴壁细胞中填充足和片状脂膜的形成。抑制PYK2后，IL-5、PAF或fMLP引起的嗜酸性粒细胞通过Transwell板的迁移被显著阻断。这些数据表明，虽然PYK2不参与β-2-整合素的黏附，但在β-2-整合素连接到内皮对抗配体后，它会引起嗜酸性粒细胞的扩散，并调节随后的趋化迁移。我们得出结论，PYK2是由β-2-整合素黏附激活的，是嗜酸性粒细胞扩散和随后的趋化迁移所必需的信号。
We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after β2-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn2+. To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by β2-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in β2-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after β2-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by β2-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.