title: FRNK inhibition of focal adhesion kinase-dependent signaling and migration in vascular smooth muscle cells.
abstract: 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.
authors: Cardiovascular Institute, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153, USA.
authors: Yevgeniya E
authors: YE
authors: Koshman
authors: Taehoon
authors: T
authors: Kim
authors: Miensheng
authors: M
authors: Chu
authors: Steven J
authors: SJ
authors: Engman
authors: Rekha
authors: R
authors: Iyengar
authors: Seth L
authors: SL
authors: Robia
authors: Allen M
authors: AM
authors: Samarel
mesh_terms: 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
chemical_list: D051419:Paxillin; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases