Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissue’s ability to contract and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause-and effect manner, regulates focal adhesion protein function and consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.

BackgroundScaffold proteins modulate cellular signaling by facilitating assembly of specific signaling pathways. However, there is at present little information if and how scaffold proteins functionally interact with each other.ResultsHere, we show that two scaffold proteins, caveolin-1 and IQGAP1, are required for phosphorylation of the actin associated pool of extracellular signal regulated kinase 1 and 2 (ERK1/2) in response to protein kinase C activation. We show by immunofluorescence and proximity ligation assays, that IQGAP1 tethers ERK1/2 to actin filaments. Moreover, siRNA experiments demonstrate that IQGAP1 is required for activation of actin-bound ERK1/2. Caveolin-1 is also necessary for phosphorylation of actin-bound ERK1/2 in response to protein kinase C, but is dispensible for ERK1/2 association with actin. Simultaneous knock down of caveolin-1 and IQGAP1 decreases total phorbol ester-induced ERK1/2 phosphorylation to the same degree as single knock down of either caveolin-1 or IQGAP1, indicating that caveolin-1 and IQGAP1 operate in the same ERK activation pathway. We further show that caveolin-1 knock down, but not IQGAP1 knock down, reduces C-Raf phosphorylation in response to phorbol ester stimulation.ConclusionsBased on our data, we suggest that caveolin-1 and IQGAP1 assemble distinct signaling modules, which are then linked in a hierarchical arrangement to generate a functional ERK1/2 activation pathway.

Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the ␣-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while ␤-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin-and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor. endosomes; microtubules; zyxin; Src DYNAMIC REMODELING of focal adhesions (FAs) and the associated actin cytoskeleton is known to be critical for cell function in proliferative and migratory cells. FA formation at the leading edge, maturation of FAs, and eventual turnover have been extensively studied (4, 61). However, the degree to which FAs in nonmigratory, fully differentiated cellular phenotypes are dynamic and the functions of those FAs embedded in tissue have yet to be fully clarified.Recent studies have demonstrated that the actin cytoskeleton in nonmigrating, contractile smooth muscle is not a totally static structure (for reviews see Refs. 24,32,60,62). In fully differentiated vascular smooth muscle (dVSM) cells, actin polymerization increases in response to the vasoconstrictor phenylephrine (PE) (33). Furthermore, it has been shown for the aorta that the actin elongation factor vasodilator-stimulated phosphoprotein (VASP) is necessary for ␣-agonist-induced actin polymerization (34). Interestingly, in airway smooth muscle, neural Wiscott-Aldrich syndrome protein activation of the actin-related protein 2/3 (Arp2...

We show here for the first time that the pro-apoptotic protein Par-4 binds to and activates myosin phosphatase (MP). During agonist stimulation, Par-4 facilitates ZIPK targeting and inhibitory phosphorylation of MP, however, phosphorylation of Par-4 is required for MP inhibition. Our model presents Par-4 as an amplifier of the MP activity range.

In smooth muscle, the extracellular signal regulated kinases 1 and 2 (ERK1/2) contextually regulate contraction or proliferation. Here, we test the hypothesis that ERK1/2 scaffolds regulate the intracellular localization and consequently, stimulus‐specific outcomes of ERK1/2 activation. SiRNA against the ERK1/2 scaffold IQGAP1 impairs phorbol ester induced ERK1/2 phosphorylation, but has no effect on basal or serum induced ERK1/2 phosphorylation in A7r5 smooth muscle cells. IP experiments show that ERK1/2 associates with both, IQGAP1 and kinase suppressor of ras‐1 under unstimulated conditions and dissociates upon serum stimulation. However, only IQGAP1 remains associated with ERK1/2 after phorbol ester stimulation. Immunofluorescence imaging and proximity ligation assays show that phorbol ester induced actin binding of phospho‐ERK1/2 is reduced after siRNA knock down of either IQGAP1 or caveolin‐1 (CaV). Interestingly, actin binding of total ERK1/2 is impaired by IQGAP1 siRNA, but not CaV siRNA. Simultaneous knock down of IQGAP1 and CaV reduces phorbol ester induced phospho‐ ERK1/2 to the same degree as IQGAP1 or CaV knock down alone, indicating that these two scaffolds act in the same ERK1/2 activation pathway. In summary, our results show that ERK1/2 scaffolds control distinct ERK1/2 pools and modulate the outcome of ERK1/2 activation in a stimulus‐specific manner. Support: HL080003, HL086655.