Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA.
Arterioscler Thromb Vasc Biol. 2011 Nov;31(11):2432-40. doi: 10.1161/ATVBAHA.111.235549.
Focal adhesion kinase-related nonkinase (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 proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling.
Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (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, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas).
We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
黏着斑激酶相关非激酶（FRNK）是黏着斑激酶（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）迁移特异性抑制剂。FRNK 在培养的 VSMC 中可抑制 FAK 和富含脯氨酸的酪氨酸激酶 2（PYK2），这两种激酶都可能参与血管重构过程中的 VSMC 浸润。
通过将绿色荧光蛋白标记的野生型（wt）FRNK 基因经腺病毒转染到球囊损伤的大鼠颈动脉，证实 FRNK 过表达可抑制体内 FAK 和 PYK2 的磷酸化及其下游信号转导。为了确定哪种激酶参与调节 VSMC 的浸润，使用腺病毒介导的特异性短发夹 RNA 表达来敲低培养的 VSMC 中的 FAK 与 PYK2，只有 FAK 短发夹 RNA 可有效降低 VSMC 的浸润。然后，使用表达非磷酸化（Tyr168Phe、Tyr232Phe 和 Tyr168，232Phe）绿色荧光蛋白-FRNK 突变体的腺病毒来研究 FRNK 酪氨酸磷酸化的作用。wtFRNK 和所有 FRNK 突变体均定位于黏着斑，但仅 Tyr168 磷酸化是 FRNK 抑制浸润所必需的。通过免疫共沉淀、全内反射荧光显微镜和光漂白后荧光恢复实验发现，阻止 Tyr168 磷酸化也增加了 FRNK 与 paxillin 的相互作用。此外，wtFRNK 与 FAK 竞争与 p130（Cas）（一种对细胞迁移至关重要的调节因子）结合，并阻止其磷酸化。然而，Tyr168Phe-FRNK 无法与 p130（Cas）结合。
我们提出了 FRNK 抑制的 3 个阶段机制：黏着斑靶向、Tyr168 磷酸化以及与 FAK 竞争 p130 结合和磷酸化，这些都是 FRNK 抑制 VSMC 浸润所必需的。
