Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle

Abstract

The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear. Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2. We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein. The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells. In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after stimulation by agonist. Antisense knockdown of SmAV in tissue inhibits both the activation of ERK1/2 and contractions stimulated by either agonist or PKC activation. This ERK1/2 signaling and contractile defect is similar to that observed in CaP knockdown experiments. In A7r5 smooth-muscle cells, PKC activation by phorbol esters induces the reorganization of endogenous, membrane-localized SmAV and microfilament-associated CaP into podosome-like structures that also contain F-actin, nonmuscle myosin IIB and ERK1/2. These results indicate that SmAV contributes to the regulation of contractility through a CaP-mediated signaling pathway, involving PKC activation and phosphorylation of ERK1/2.

Smooth Muscle Archvillin Is an ERK Scaffolding Protein

“…Previously, PE stimulation has been shown to target ERK to the cell surface (8), but depolarization leads to a homogeneous cellular distribution of ERK (20). Interestingly, both SmAV and ERK translocate to the membrane upon PE stimulation at the same time point (4 min) in isolated ferret aorta cells, as has been shown previously (7,8). The stimulus-specific nature of N-SmAV1 binding to ERK signaling partners could serve to spatially sequester the ␣-agonist-activated ERK pathway from the depolarization-activated ERK pathway and provide a mechanism whereby ERK in the intracellular environment chooses the signaling pathway to follow in response to a specific agonist.…”

“…SmAV Interacts with Two ERK Domains-We have previously predicted, based on sequence analysis, that SmAV contains 2 ERK-binding domains (7), one at residue 219 and a second at residue 774. To determine whether either or both of these predicted sites bind ERK, two N-terminal fragments were expressed as recombinant fragments with N-terminal chitinbinding domain tags.…”

“…Scaffolds have been defined as proteins, generally with no applicable enzymatic activity "that interact with a signaling pathway to create a functional signaling module and to control the specificity of signal transduction" (5). We have previously shown that SmAV knockdown in vascular smooth muscle tissue inhibits ERK activation and contraction induced by the ␣-agonist PE (7). We demonstrate here that N-terminal SmAV sequence (N-SmAV-1) associates with ERK, MEK, Raf, and 14-3-3, all known members of a classical ERK signaling pathway, in an agonist-and pathwayspecific manner.…”

“…Leinweber et al (23) showed that the ERK-binding domain of calponin is the N-terminal calponin homology domain, which Gangopadhyay et al (7) showed is the domain of calponin that binds the C-terminal end of SmAV. These past studies can be combined with the work presented here in a model shown in Phosphorylation of caldesmon makes the actin available for the interaction with myosin, which in turn increases contractility.…”

“…ERK Phosphorylates N-SmAV1 but Not N-SmAV2-Analysis of the SmAV sequence predicts for a proline-directed kinase (such as ERK) phosphorylation site to be present adjacent to the predicted ERK-binding site in SmAV1 (7). Thus, the question arises as to whether the activated ERK1/2 bound to SmAV1 might use SmAV itself as a substrate.…”