Critical role of vimentin phosphorylation at Ser-56 by p21-activated kinase in vimentin cytoskeleton signaling.
Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and mitosis. In this study, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of p21-activated kinase (PAK) activation). Treatment with PAK led to disassembly of wild-type (but not mutant S56A) vimentin filaments as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS; an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of mutant S56A vimentin in cells inhibited the increase in phosphorylation at Ser-56 and in the ratios of soluble to insoluble vimentin (an index of vimentin disassembly) and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation compared with cells expressing wild-type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing the S56A mutant. Expression of mutant S56A vimentin depressed PAK phosphorylation at Thr-423 induced by 5-HT. Expression of the S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.
Li
Qing-Fen
QF
Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
Spinelli
Amy M
AM
Wang
Ruping
R
Anfinogenova
Yana
Y
Singer
Harold A
HA
Tang
Dale D
DD
D000199:Actins / Q000378:metabolism; D000818:Animals; D050719:Crk-Associated Substrate Protein / Q000378:metabolism; D003599:Cytoskeleton; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D018490:Serotonin Agents / Q000494:pharmacology; D015398:Signal Transduction; D014132:Trachea / Q000187:drug effects / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases
D000199:Actins; D050719:Crk-Associated Substrate Protein; D018490:Serotonin Agents; D014746:Vimentin; D010768:Phosphoserine; D012701:Serotonin; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases