Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages
Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages
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DOI：
10.1242/jcs.051755
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发表时间：
2009-11-01
影响因子：
4
通讯作者：
Cox, Dianne
中科院分区：
文献类型：
--
作者：
Dovas, Athanassios;Gevrey, Jean-Claude;Cox, Dianne
Podosomes是一种能够降解基质的粘附结构，与细胞趋化和侵入组织的能力有关。Wiskott-Aldrich综合征蛋白（WASp）是RhoGTPase Cdc42和Src家族激酶底物的效应蛋白，调控巨噬细胞足体的形成。在这项研究中，我们通过使用TIRF-FRET显微镜证明WASp在足质体中具有活性。药理学和RNA干扰方法表明，足小体的形成和功能需要持续的WASp活性。利用点突变进行的援救实验表明，在足体形成过程中，Cdc42与WASp结合是绝对必要的。虽然酪氨酸磷酸化并非足小体形成的绝对必要条件，但磷酸化确实调节足小体成核速率和肌动蛋白丝稳定性。重要的是，WASp酪氨酸磷酸化不会改变WASp的激活，相反，磷酸化似乎对限制WASp对足小体的活性很重要。此外，细胞的基质降解能力需要WASp磷酸化。在缺乏内源性WASp的情况下，对CSF-1的趋化反应也减弱了，这两种酪氨酸突变都无法挽救WASp。这些结果表明，酪氨酸磷酸化的作用比简单地调节WASp活性更为复杂，并且表明足质体动力学和巨噬细胞迁移之间存在联系。
Podosomes, adhesion structures capable of matrix degradation, have been linked with the ability of cells to perform chemotaxis and invade tissues. Wiskott-Aldrich Syndrome protein (WASp), an effector of the RhoGTPase Cdc42 and a Src family kinase substrate, regulates macrophage podosome formation. In this study, we demonstrate that WASp is active in podosomes by using TIRF-FRET microscopy. Pharmacological and RNA interference approaches suggested that continuous WASp activity is required for podosome formation and function. Rescue experiments using point mutations demonstrate an absolute requirement for Cdc42 binding to WASp in podosome formation. Although tyrosine phosphorylation was not absolutely required for podosome formation, phosphorylation did regulate the rate of podosome nucleation and actin filament stability. Importantly, WASp tyrosine phosphorylation does not alter WASp activation, instead phosphorylation appears to be important for the restriction of WASp activity to podosomes. In addition, the matrix-degrading ability of cells requires WASp phosphorylation. Chemotactic responses to CSF-1 were also attenuated in the absence of endogenous WASp, which could not be rescued with either tyrosine mutation. These results suggest a more complex role for tyrosine phosphorylation than simply in the regulation of WASp activity, and suggest a link between podosome dynamics and macrophage migration.