Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant
Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant
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DOI：
10.1189/jlb.0206107
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发表时间：
2007-11-01
影响因子：
5.5
通讯作者：
Jones, Samuel L.
中科院分区：
文献类型：
--
作者：
Eckert, Rachael E.;Jones, Samuel L.
血管舒张刺激磷蛋白（VASP）是一种cAMP依赖性蛋白激酶A（PKA）底物，它将细胞信号传导与细胞骨架组织和细胞运动联系起来。VASP被PKA在丝氨酸157（Ser 157）上磷酸化，这是VASP在血小板粘附和成纤维细胞运动中的功能所需的。我们的假设是PKA通过VASP Ser 157磷酸化调节中性粒细胞迁移。本研究的目的是表征趋化因子刺激的中性粒细胞中VASP Ser 157磷酸化。fMLF、IL-8、白三烯B-4或血小板活化因子刺激导致VASP Ser 157磷酸化最初增加，30 s时最大，10 min后恢复至基线Ser 157磷酸化。相反，用非趋化因子、促炎细胞因子TNF-α刺激不影响Ser 157磷酸化。fMLF诱导的VASP Ser 157磷酸化水平的动力学与fMLF刺激的中性粒细胞中F-肌动蛋白水平的倍数变化的动力学密切匹配。用PKA抑制剂H89和腺苷酸环化酶抑制剂SQ 22536预处理可消除fMLF诱导的Ser 157磷酸化。相比之下，fMLF诱导的Ser 157磷酸化不受PKC抑制剂calphostin和staurosporine、PKG抑制剂Rp-8-pCPT-cGMP和KT 5823以及钙调蛋白依赖性蛋白激酶11抑制剂KN-62的影响。用EDTA或抗β 2-整联蛋白抗体IB 4抑制粘附并不改变fMLF诱导的VASP磷酸化或去磷酸化。这些数据表明，人中性粒细胞的化学引诱物刺激诱导快速和短暂的PKA依赖性VASP Ser 157磷酸化。粘附似乎不是化学引诱剂刺激的中性粒细胞中VASP Ser 157磷酸化状态的重要调节剂。
Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hypothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The objective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. fMLF, IL-8, leukotriene B-4, or platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoattractant, proinflammatory cytokine TNF-alpha did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation levels closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT-cGMP and KT5823, and the calmodulin-dependent protein kinase 11 inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-beta 2-integrin antibody IB4 did not alter fMLF-induced VASP phosphorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhesion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils.