Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding.
Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding.
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DOI：
10.1161/atvbaha.111.235549
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发表时间：
2011-11
期刊：
影响因子：
--
通讯作者：
Samarel AM
中科院分区：
文献类型：
--
作者：
Koshman YE;Chu M;Engman SJ;Kim T;Iyengar R;Robia SL;Samarel AM
FRANK是粘着斑激酶(FAK)的C末端结构域，是一种酪氨酸磷酸化的、血管平滑肌细胞(VSMC)特异性的细胞迁移抑制因子。FRNK对培养的VSMC的FAK和PYK2均有抑制作用，二者可能参与了血管重塑过程中VSMC的侵袭。腺病毒介导的GFP标记的野生型(Wt)frnk基因转移到球囊损伤的大鼠颈动脉证实，frnk过表达在体内抑制了FAK和PYK2的磷酸化及其下游信号转导。为了确定哪一种激酶参与了VSMC侵袭的调控，用腺病毒介导的特定shRNAs的表达来“敲除”培养的VSMC中的FAK和PYK2，但只有FAK shRNA能有效地减少VSMC的侵袭。然后用表达不可磷酸化的(Y168F-、Y232F-和Y168,232F-)GFP-frnk突变体的腺病毒检测frnk酪氨酸磷酸化的作用。WtFRNK和所有的frnk突变体都定位于FAs，但frnk只需要Y168的磷酸化就能抑制侵袭。通过免疫共沉淀、全内反射荧光(TIRF)显微镜和光漂白后荧光恢复(FRAP)检测，阻止Y168磷酸化也增加了frnk-paxlin的相互作用。此外，wtFRNK与FAK竞争结合p130Cas(一个至关重要的细胞迁移调节因子)，并阻止其磷酸化。然而，Y168F-frnk不能结合p130Cas。我们提出了FRNK抑制的三阶段机制--FA靶向、Y168磷酸化以及与FAK竞争p130Cas结合和磷酸化，这些都是FRNK抑制VSMC侵袭所必需的。
FRNK, the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and PYK2 in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. Adenoviral-mediated gene transfer of GFP-tagged, wildtype (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenoviral-mediated expression of specific shRNAs were used to “knock down” FAK vs. PYK2 in cultured VSMCs, but only FAK shRNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Y168F-, Y232F-, and Y168,232F-) GFP-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Y168 phosphorylation was required for FRNK to inhibit invasion. Preventing Y168 phosphorylation also increased FRNK-paxillin interaction, as determined by co-immunoprecipitation, total internal reflection fluorescence (TIRF)-microscopy, and fluorescence recovery after photobleaching (FRAP). Furthermore, wtFRNK competed with FAK for binding to p130Cas (a critically important regulator of cell migration), and prevented its phosphorylation. However, Y168F-FRNK was unable to bind p130Cas. We propose a 3-stage mechanism for FRNK inhibition – FA targeting, Y168 phosphorylation, and competition with FAK for p130Cas binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.