Role of FRNK tyrosine phosphorylation in vascular smooth muscle spreading and migration.
OBJECTIVE
Focal adhesion kinase (FAK) and its autonomously expressed, C-terminal inhibitor FAK-related non-kinase (FRNK), are important regulators of vascular smooth muscle cell (VSMC) spreading and migration. However, the mechanisms of FRNK-mediated inhibition of FAK-dependent signalling are not fully defined. The aim of this study was to determine the potential role of FRNK tyrosine phosphorylation in regulating these processes.


RESULTS
Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in alpha-smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation.


CONCLUSIONS
Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.
Koshman
Yevgeniya E
YE
The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA.
Engman
Steven J
SJ
Kim
Taehoon
T
Iyengar
Rekha
R
Henderson
Kyle K
KK
Samarel
Allen M
AM
D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000502:physiology; D022001:Focal Adhesions; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases