FRNK inhibition of focal adhesion kinase-dependent signaling and migration in vascular smooth muscle cells.
OBJECTIVE
To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation.


RESULTS
Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase-related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein-tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II-stimulated focal adhesion kinase, paxillin, and extracellular signal-regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK-expressing VSMCs. Angiotensin II-induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2.


CONCLUSIONS
FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.
Koshman
Yevgeniya E
YE
Cardiovascular Institute, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153, USA.
Kim
Taehoon
T
Chu
Miensheng
M
Engman
Steven J
SJ
Iyengar
Rekha
R
Robia
Seth L
SL
Samarel
Allen M
AM
D000818:Animals; D002465:Cell Movement / Q000502:physiology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D015398:Signal Transduction
D051419:Paxillin; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases