bPAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin.
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.
Lee
Chan Soo
CS
Department of Biochemistry, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju 361-763 Korea.
Kim
Kyung Yong
KY
Im
Jae Bin
JB
Choi
Jae Woon
JW
Kim
Hyong Kyu
HK
Park
Jeong Soo
JS
Shin
Eun Young
EY
Kim
Seung Ryul
SR
Kim
Eung Gook
EG
D008841:Actin Cytoskeleton / Q000502:physiology*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018797:Cell Cycle Proteins / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000378:metabolism; D018604:Epitope Mapping; D020662:Guanine Nucleotide Exchange Factors / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D047468:Immunoprecipitation; D051379:Mice; D017434:Protein Structure, Tertiary; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors
D000199:Actins; D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors