FRNK Inhibits VSMC Invasion by Tyrosine Phosphorylation at Y168 and Competition for FAK Binding

FRNK inhibits FAK and PYK2 signaling in VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. To determine which kinase is responsible, shRNAs specific for each kinase were used to knock down FAK vs. PYK2 in cultured VSMCs. Only FAK shRNA was effective in reducing VSMC invasion in a 3D Boyden chamber assay. We previously showed that FRNK undergoes Y168 and Y232 phosphorylation during vascular injury in vivo, and angiotensin II (AngII) stimulation in cultured VSMCs. We now examine the functional significance of FRNK‐Y168 and ‐Y232 phosphorylation by overexpressing GFP‐tagged, wildtype (wt) and nonphosphorylatable FRNK (Y168F‐, Y232F‐, and Y168,232F‐FRNK). wtFRNK and FRNK mutants all localized to FAs, as detected by TIRF‐microscopy of living VSMCs. Only phosphorylation of Y168 (but not Y232) was required for FRNK to inhibit VSMC invasion. FRAP was then used to analyze whether phosphorylation affected FRNK binding kinetics to FAs. K FRAP was significantly reduced in Y168F and Y168,232F mutants, indicating that their binding affinity to FA proteins was increased. However, AngII had no further effect on K FRAP for either wtFRNK or Y168F‐FRNK. We conclude that both FRNK‐Y168 phosphorylation and FRNK targeting to FAs are required for FRNK to inhibit VSMC invasion. Supported by the Falk Medical Research Trust and an AHA‐Midwest Postdoctoral Fellowship.