Novel Mechanisms of FRNK Inhibition of FAK in Vascular Smooth Muscle Cells

Abstract

The nonreceptor protein tyrosine kinase focal adhesion kinase (FAK) is an important regulator of growth and migration in smooth muscle cells that acts as a kinase and a scaffolding protein. FAK-related nonkinase (FRNK) is an endogenously expressed truncated form of FAK that inhibits FAK signaling through an unknown mechanism. Traditionally, FRNK has been thought to inhibit FAK by displacing it from focal adhesion binding sites. We propose an additional parallel mechanism wherein FRNK binds directly to FAK, inhibiting downstream signaling. Initial co-immunoprecipitation experiments suggested FAK and FRNK interact. We quantified fluorescence resonance energy transfer (FRET) from FAK to FRNK using acceptor photobleaching and fluorescence lifetime measurements. Both approaches used total internal reflection fluorescence (TIRF) microscopy for selective illumination. Acceptor photo-bleaching experiments indicated 5.2±1.2% FRET between CER-FRNK and YFP-FAK. Fluorescence lifetime FRET experiments revealed 8.6±0.9% FRET between GFP-FAK and mCherry-FRNK. Furthermore, we show that FAK and FRNK localize to specific locations within focal adhesions. FRNK appears to be localized preferentially towards the proximal side of the focal adhesion. We hypothesize that this polarization is induced by strain on the focal adhesion from the attached actin stress fiber. Lastly, we have identified a phosphorylation site on FRNK, serine at position 217, that appears to regulate FRNK inhibition. A phosphomimetic mutant with serine 217 mutated to aspartic acid reduced FAK Y397 (activation site) phosphorylation by approximately 50% compared to wild type. The phosphomimetic mutant tagged with mCherry exhibited reduced FRET with GFP-FAK (5.4 ± 0.8) compared to wild type FRNK (8.6 ± 0.9%) suggesting decreased FAK binding or an altered conformation of the FAK/FRNK regulatory complex. We conclude that FRNK regulates FAK through a direct inhibitory interaction, and the inhibitory potency of FRNK is modulated by phosphorylation of serine 217.