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The intermediate filament framework is one of the three cytoskeletal systems in mammalian cells. Its well spread filamentous structure from the nucleus to the plasma membrane is believed to provide protection against various mechanical stresses (1, 2). Intermediate filaments also undergo disassembly/assembly and spatial reorganization in cells in response to external stimulation and during mitosis. The dynamic property of the intermediate filament system plays a fundamental role in mediating changes in cell shape, division, and migration; signaling molecule distribution; and smooth muscle force development (1, 3-7).The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation. Vimentin is the most abundant intermediate filament protein in various cell types, including smooth muscle cells (2,5,6). Vimentin phosphorylation in association with vimentin disassembly and spatial reorganization occurs during mitosis or in response to extracellular stimulation (8, 9). In cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation at Ser-56 concurrently with vimentin partial disassembly and spatial reorientation (6).The disassembly and spatial reorganization of the vimentin network may regulate the translocation of certain molecules (7, 10, 11). The adapter protein p130 Crk-associated substrate (CAS) 2 has been shown to participate in the signaling processes that regulate smooth muscle contraction and cell migration (12-14). Our recent study has suggested that vimentin phosphorylation and disassembly are related to CAS redistribution during contractile activation of smooth muscle (10). In addition, external stress initiates Rho kinase redistribution associated with vimentin depolymerization in fibroblasts and the translocation of Ca 2ϩ /calmodulin-dependent protein kinase II in differentiated smooth muscle cells, which may be an important event for cell signaling (7, 11).p21-activated kinase (PAK) may be an upstream regulator of the vimentin network (6, 9). In cultured smooth muscle cells, agonist-mediated vimentin phosphorylation at Ser-56 and spatial reorientation of the vimentin network are inhibited by silencing of PAK1, a dominant isoform in smooth muscle (6,15). Additionally, PAK has been implicated in modulating smooth muscle contraction; introduction of an active PAK isoform into smooth muscle potentiates force development at constant intracellular calcium (16). Expression of an inactive PAK1 mutant attenuates migration of cultured smooth muscle cells in response to platelet-derived growth factor (15).In response to external stimulation, PAK undergoes autophosphorylation at Thr-423, which increases PAK activity for substrates (17,18). In addition to the small GTPases Cdc42 and Rac1, the activity of PAK may be regulated by the paxillin kinase linker/PIX (PAK-interacting exchange factor; guanine nucleotide exchange factor) (19 -21). CAS has been shown to interact with the paxillin kinase linker/PIX via CrkII and paxillin (21-* This work was ...

Abstract-The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation. Key Words: tyrosine kinase Ⅲ actin cytoskeleton Ⅲ contraction Ⅲ vascular smooth muscle Ⅲ adapter protein A ctin cytoskeleton remodeling has recently emerged as an important cellular process mediating smooth muscle contraction. 1-7 A pool of globular actin (G-actin) is stimulated onto filamentous actin (F-actin) in a variety of smooth muscle cells and tissues in response to agonist stimulation. Blockage of actin polymerization by the inhibitors cytochalasin and latrunculin attenuates active force on activation with contractile stimuli whereas myosin regulatory light chain phosphorylation (MRLCP) is not disrupted. 5,8 -10 These studies suggest that actin dynamics and MRLCP are independently regulated, and that both dynamic changes in the actin cytoskeleton and myosin activation are required for force development during contractile stimulation of smooth muscle. 4,8 -11 However, the mechanisms that regulate the actin cytoskeleton in smooth muscle are not completely understood.The tyrosine phosphorylated protein Crk-associated substrate (CAS) has been implicated in the modulation of the actin cytoskeleton in smooth muscle cells as well as in nonmuscle cells including COS-7 cells and NIH3T3 cells. 9,12-14 Downregulation of CAS by antisense dramatically attenu...

Raf-1 is a serine/threonine protein kinase that has an essential role in cell proliferation. The mechanisms that regulate Raf-1 in airway smooth muscle are not well understood. In this study, treatment with platelet-derived growth factor (PDGF) induced spatial redistribution of Raf-1 from the cytoplasm to the periphery of human airway smooth muscle cells. Moreover, a pool of Raf-1 was found in F-actin of human airway smooth muscle cells. Activation with PDGF led to an increase in the association of Raf-1 with cytoskeletal actin. Treatment of cells with the actin polymerization inhibitor latrunculin A (LAT-A), but not the microtubule depolymerizer nocodazole, inhibited the interaction of Raf-1 with actin in response to PDGF activation. Because abelson tyrosine kinase (Abl) is known to specifically regulate actin dynamics in smooth muscle, the role of Abl in modulating the coupling of Raf-1 with actin was also evaluated. Abl knockdown by RNA interference attenuated the association of Raf-1 with actin, which is recovered by Abl rescue. Treatment with LAT-A, but not nocodazole, inhibited the spatial redistribution of Raf-1 during PDGF activation. However, treatment with both LAT-A and nocodazole attenuated smooth muscle cell proliferation. Finally, Abl knockdown attenuated the redistribution of Raf-1 and cell proliferation, which were restored by Abl reexpression. The results suggest a novel mechanism that the interaction of Raf-1 with cytoskeletal actin is critical for Raf-1 redistribution and airway smooth muscle cell proliferation during activation with the growth factor.

The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, stimulation of tracheal smooth muscle strips with acetylcholine (ACh) resulted in the increase in ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly) in association with force development. Activation with ACh also induced vimentin phosphorylation at Ser-56 as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips upon contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The decrease in distribution of CAS in cytoskeletal vimentin elicited by ACh was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble vimentin to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated the dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during acetylcholine activation of tracheal smooth muscle tissues.