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Focal Adhesion Kinase forms Inhibitory Complexes with its Endogenous Inhibitor
Author(s) -
Zak Taylor J,
Robia Seth L,
Samarel Allen M
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.880.6
Subject(s) - focal adhesion , phosphorylation , kinase , microbiology and biotechnology , biology , serine , signal transduction , ptk2 , cancer research , protein kinase a , mitogen activated protein kinase kinase
Focal adhesion kinase related non‐kinase (FRNK) is an inhibitor of focal adhesion kinase (FAK)‐dependent cell growth, survival, and migration pathways. In vascular smooth muscle cells, both FAK and FRNK are upregulated after vascular injury, suggesting balanced stimulation and inhibition of growth during recovery. Better understanding of the molecular mechanisms of FRNK inhibition of FAK are needed to understand vascular smooth muscle cell response to injury. Here we propose a novel mechanism by which FRNK can inhibit FAK: direct binding of FRNK to FAK forming an inhibitory complex within focal adhesions. This hypothesis is supported by co‐immunoprecipitation of FAK and FRNK. In addition, fluorescence resonance energy transfer (FRET) measurements suggest that FRNK binds directly to FAK within focal adhesions of live vascular smooth muscle cells. We hypothesized that this interaction might be regulated by serine phosphorylation of FRNK S217, a site we found to be basally highly phosphorylated at S217 by extracellular signal–regulated kinase 1/2 (ERK). To determine the significance of S217 phosphorylation, we generated a non‐phosphorylatable mutant FRNK by mutating serine 217 to alanine (S217A). S217A‐FRNK exhibited significantly higher amounts of FRET compared to wild type FRNK, suggesting increased interaction with FAK. Progressive acceptor photobleaching analysis revealed that the FRNK‐FAK complex consisted of FRNK and multiple FAK proteins in a high order hetero‐oligomer. The functional significance of the S217 phosphorylation site was suggested by S217A FRNK's more potent inhibition of FAK Y397 phosphorylation compared to wild type FRNK. FRNK S217A more potently decreased cell growth / survival compared to wild type. Specifically, S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type FRNK, as determined by annexin staining and flow cytometry. We hypothesize that upregulation of FRNK after vascular injury results in the formation of a FRNK‐FAK inhibitory complex that serves to attenuate injury related growth and that phosphorylation of FRNK at S217 by ERK decreases the inhibitory FRNK‐FAK complex formation, allowing the cell to return to a normal resting state once healing has completed. Support or Funding Information NIH: R01‐HL092321 and R01‐HL106189 to S.L.R., P01‐HL62426 to A.M.S., and F32‐HL096143 to Y.E.K.