Actopaxin is phosphorylated during mitosis and is a substrate for cyclin B1/cdc2 kinase.
Prior to cell division, normal adherent cells adopt a round morphology that is associated with a loss of actin stress fibres and disassembly of focal adhesions. In this study, we investigate the mitotic phosphorylation of the recently described paxillin and actin-binding focal-adhesion protein actopaxin [Nikolopoulos and Turner (2000) J. Cell Biol. 151, 1435-1448]. Actopaxin is comprised of an N-terminus containing six putative cdc2 phosphorylation sites and a C-terminus consisting of tandem calponin homology domains. Here we show that the N-terminus of actopaxin is phosphorylated by cyclin B1/cdc2 kinase in vitro and that this region of actopaxin precipitates cdc2 kinase activity from mitotic lysates. Actopaxin exhibits reduced electrophoretic mobility during mitosis that is dependent on phosphorylation within the first two consensus cdc2 phosphorylation sites. Finally, as cells progress from mitosis to G(1) there is an adhesion-independent dephosphorylation of actopaxin, suggesting that actopaxin dephosphorylation precedes cell spreading and the reformation of focal adhesions. Taken together, these results suggest a role for cyclin B1/cdc2-dependent phosphorylation of actopaxin in regulating actin cytoskeleton reorganization during cell division.
Curtis
Michael
M
Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210, U.S.A.
Nikolopoulos
Sotiris N
SN
Turner
Christopher E
CE
D000185:Actinin; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D016466:CHO Cells; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D000096985:Calponins
D000185:Actinin; D000199:Actins; D016203:CDC2 Protein Kinase; D019926:Cyclin B; D056744:Cyclin B1; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D012694:Serine; C534699:CCNB1 protein, human; C534701:Ccnb1 protein, rat; C486590:PARVA protein, human; D000096985:Calponins