title: Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA.
authors.forename: Yevgeniya E
authors.initials: YE
authors.lastname: Koshman
authors.forename: Miensheng
authors.initials: M
authors.lastname: Chu
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Engman
authors.forename: Taehoon
authors.initials: T
authors.lastname: Kim
authors.forename: Rekha
authors.initials: R
authors.lastname: Iyengar
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
authors.forename: Allen M
authors.initials: AM
authors.lastname: Samarel
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002339:Carotid Arteries / Q000378:metabolism / Q000473:pathology; D020212:Carotid Artery Injuries / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D002404:Catheterization / Q000009:adverse effects; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D023421:Models, Animal; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats
chemical_list: D050719:Crk-Associated Substrate Protein; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases