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