Exp Mol Med.  2004 Dec;36(6):582-587.

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

• Lee CS
• Kim KY
• Im JB
• Choi JW
• Kim HK
• Park JS
• Shin EY
• Kim SR
• Kim EG

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

Keyword

actin; beta PAK; monoclonal antibody; neurite outgrowth

MeSH Terms

Actins/*metabolism
Animals
Antibodies, Monoclonal/immunology
Cell Cycle Proteins/immunology/metabolism/*physiology
Cell Line, Tumor
Cytoskeletal Proteins/metabolism
Epitope Mapping
Guanine Nucleotide Exchange Factors/immunology/metabolism/*physiology
Immunoprecipitation
Mice
Microfilaments/*physiology
Protein Structure, Tertiary
Rats
Research Support, Non-U.S. Gov't