A new signaling paradigm for serotonin: use of Crk-associated substrate in arterial contraction

Abstract

A new signaling paradigm for serotonin: use of Crk-associated substrate in arterial contraction. Am J Physiol Heart Circ Physiol 291: H2857-H2863, 2006. First published July 21, 2006 doi:10.1152/ajpheart.00229.2006.-Crkassociated substrate (CAS), a 130-kDa adaptor protein, was discovered as a tyrosine kinase substrate of Src that was important to cellular motility and actin filament formation. As the tyrosine kinase Src is utilized by the 5-hydroxytryptamine (5-HT)2A receptor in arterial contraction, we tested the hypothesis that CAS was integral to 5-HT2A receptor-mediated vasoconstriction. Rat thoracic aorta was used as a model of the arterial 5-HT2A receptor. Western and immunohistochemistry analyses validated the presence of CAS in the aorta, and tissue bath experiments demonstrated reduction of contraction to 5-HT (13.5 Ϯ 5% control maximum) and the 5-HT2 receptor agonist ␣-methyl-5-HT (6 Ϯ 2% maximum) by latrunculin B (10 Ϫ6 mol/l), an actin disruptor. In aorta contracted with 5-HT (10 Ϫ5 mol/l), tyrosine phosphorylation (Tyr410) of CAS was significantly increased (ϳ225%), and both contraction and CAS phosphorylation were reduced by the 5-HT2A/2C receptor antagonist ketanserin (3 ϫ 10 Ϫ8 mol/l). Src is one candidate for 5-HT-stimulated CAS tyrosyl-phosphorylation as 5-HT promoted interaction of Src and CAS in coimmunoprecipitation experiments, and the Src tyrosine kinase inhibitor PP1 (10 Ϫ5 mol/l) abolished 5-HT-induced tyrosyl-phosphorylation of CAS and reduced 5-HT-and ␣-methyl-5-HT-induced contraction. Antisense oligodeoxynucleotides delivered to the aorta reduced CAS expression (33% control) and arterial contraction to ␣-methyl-5-HT (45% of control), independent of changes in myosin light chain phosphorylation. These data are the first to implicate CAS in the signal transduction of 5-HT. 5-hydroxytryptamine; antisense oligodeoxynucleotides; signal transduction; Src THE ACTIVATION of protein kinases in phosphorylation cascades is critical to signal transduction. This process is facilitated by adaptor proteins that contain Src homology 2 (SH2) and Src homology 3 (SH3) protein regions that allow for docking of other proteins and facilitation of protein-protein interactions. One such adapter protein is Crk-associated substrate or CAS (also known as p130 or p130 CAS). This 130-kDa protein has been recognized as a substrate for the tyrosine kinase Crk (23), and CAS is highly tyrosine phosphorylated in response to activation of Crk or Src (25,26). Docking of and tyrosylphosphorylation by these proteins, as well as other regulators, including focal adhesion kinase (FAK) and protein tyrosine phosphatase 1B (PTP1B), enables CAS to participate in important physiological events, including cell migration (14, 18, 27), mitosis (30), adhesion (42), and apoptosis (48).The involvement of CAS in contraction is logical because CAS is essential for actin filament reorganization (12, 38). CAS is a regulator of the actin-associated protein profilin (14), which is necessary for actin-filament polymerization in smooth muscl...

Cdc42GAP, reactive oxygen species, and the vimentin network

“…Vimentin consists of an NH 2 -terminal head domain containing several phosphorylation sites, a central ␣-helical rod domain (forming the backbone of filaments) and a COOHterminal tail domain (16,26 (4,24,40,46). In smooth muscle cells/tissues, disassembly of the vimentin system may regulate the translocation of Crk-associated substrate (CAS) and Ca 2ϩ /calmodulin-dependent protein kinase II (CamKII), which may participate in the cellular processes that control force development (1,21,23,27,29,35,37,45). In this report, the expression of Cdc42GAP, but not its inactive mutant, inhibits cell contraction as assessed by the collagen gel constriction assay.…”

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

“…The pharmacologic agents latrunculin and cytochalasin, which inhibit actin polymerization by sequestering G-actin monomers and by capping actin filaments, respectively (17,32,35), have been widely employed in a variety of smooth muscle tissue and cell types to evaluate the effects of inhibiting actin polymerization on contractile responses to agonist stimulation. Studies of airway smooth muscle (6,43,84,115,131,140), vascular smooth muscle (1,25,27,28,92,93,104,107,112,138), and uterine (112) and intestinal smooth muscle (81,91) have all shown that the short-term exposure of smooth muscle tissues to inhibitors of actin polymerization causes a profound suppression of tension development and an inhibition of shortening or constriction. Additional evidence that actin polymerization plays a critical role in the process of mechanical tension development in smooth muscle comes from studies showing that molecular constructs or peptides that disrupt specific steps in the actin polymerization process also inhibit tension development in smooth muscle tissues in response to contractile stimuli (7,123,127,128,143,144).…”

The Plastic Nature of the Vascular Wall: A Continuum of Remodeling Events Contributing to Control of Arteriolar Diameter and Structure

“…Indeed, as in airway smooth muscle, studies investigating the involvement of actin remodeling in vasoconstriction indicate that inhibition of actin polymerization blocks the development of full contractile force of both conduit and small resistance arteries (2,36,43,44,123,124,137,142,148,176). However, in contrast to tracheal smooth muscle, inhibition of actin polymerization appears to interfere with vasoconstriction in a stimulus-and time-specific manner.…”