Critical Role of Vimentin Phosphorylation at Ser-56 by p21-activated Kinase in Vimentin Cytoskeleton Signaling

Qing-Fen Li,Amy M Spinelli,Ruping Wang,Yana Anfinogenova,Harold A Singer,Dale D Tang

doi:10.1074/jbc.m607715200

Qing-Fen Li, Amy M Spinelli + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m607715200

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Abstract

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.

Highlights

The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation
Incubation of the mutant with p21-activated kinase (PAK) did not trigger the disassembly of mutant S56A vimentin filaments (Fig. 3d). These results suggest that vimentin phosphorylation at Ser-56 by PAK leads directly to vimentin filament disassembly
The results from this study show, first, that PAK is able to directly initiate vimentin disassembly associated with Ser-56 phosphorylation

Summary

Introduction

The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation. In cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation at Ser-56 concurrently with vimentin partial disassembly and spatial reorientation [6]. The adapter protein p130 Crk-associated substrate (CAS) has been shown to participate in the signaling processes that regulate smooth muscle contraction and cell migration [12,13,14]. 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]. The aim of this study was to test the hypothesis that vimentin phosphorylation at Ser-56 may play a critical role in regulating PAK activation and the spatial reorientation of the vimentin network. Expression of a non-phosphorylatable vimentin mutant (S56A) attenuates the increase in PAK activation and the structural rearrangement of vimentin filaments upon agonist stimulation

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