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

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 βPIK. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of βPIK were generated and characterized. N-C6 Mab detected βPIK 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 βPIK. Using C-A3 Mab possible βPIK interaction with actin in PC12 cells was examined. βPIK Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecpitate βPIK. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of βPIK in the cell lysates. These results suggest that βPIK may not interact with soluble actin but with polymerized F-actin and revealed that βPIK constitutes a functional complex with actin. These data indicate real usefulness of the βPIK Mab in the study of βPIK role(s) in regulation of actin cyoskeleton.