Mutant Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and inhibits cytokinesis.
Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division of Xenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.
Yamashiro
S
S
Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Labs, Busch Campus, Piscataway, New Jersey 08855, USA. yamashiro@mbcl.rutgers.edu
Chern
H
H
Yamakita
Y
Y
Matsumura
F
F
D008841:Actin Cytoskeleton / Q000187:drug effects; D000818:Animals; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D016466:CHO Cells; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects*; D006224:Cricetinae; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects; D004625:Embryo, Nonmammalian; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000187:drug effects*; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D014162:Transfection; D014981:Xenopus
D002148:Calmodulin-Binding Proteins; D016203:CDC2 Protein Kinase