β PAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin

Chan-Soo Lee,Eun-Young Shin,Jae-Bin Im,Jae-Woon Choi,Kyung-Yong Kim,Hyong-Kyu Kim,Eung-Gook Kim,Seung Ryul Kim,Jeong-Soo Park

doi:10.1038/emm.2004.75

Chan-Soo Lee, Eun-Young Shin + Show 7 more

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https://doi.org/10.1038/emm.2004.75

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Abstract

p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.

Highlights

Small G proteins of the Rho family are critical regulators in a wide variety of cellular processes such as actin dynamics, gene transcription, cell-cycle progression and cell adhesion (Hall A, 1998). They convert between the inactive GDP-bound and the active GTP-bound state, which is tightly coordinated by a guanine nucleotide exchange factor (GEF), a GDPdissociation inhibitor (GDI), and a GTPase activating protein (GAP)
Accumulating evidence indicates that RhoGDI is first dissociated from the Rho GTPase complex, and GDP-GTP exchange by a GEF occurs
DerMardirossian et al, (2004) elegantly showed that this dissociation is mediated by p21-activated kinase (Pak) through phosphorylation of RhoGDI at Ser101 and Ser174

Summary

Introduction

Small G proteins of the Rho family are critical regulators in a wide variety of cellular processes such as actin dynamics, gene transcription, cell-cycle progression and cell adhesion (Hall A, 1998) They convert between the inactive GDP-bound and the active GTP-bound state, which is tightly coordinated by a guanine nucleotide exchange factor (GEF), a GDPdissociation inhibitor (GDI), and a GTPase activating protein (GAP). Because of this interaction, when stimulated by growth factors such as basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), PAK is capable of phosphorylating βPIX at Ser525/Thr526 in the GIT1-binding domain (GBD) (Shin et al, 2002) This results in activation of βPIX for its downstream Rac1/Cdc GTPases (Shin et al, 2004). Using these antibodies we further showed that PIX interacts with actin

Materials and Methods

Findings

PIX 495