Characterization of mitotically phosphorylated caldesmon.
Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.
Yamakita
Y
Y
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1059.
Yamashiro
S
S
Matsumura
F
F
D000199:Actins / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D008938:Mitosis; D009218:Myosins / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D000199:Actins; D002148:Calmodulin-Binding Proteins; D011494:Protein Kinases; C056959:caldesmon kinase; D016203:CDC2 Protein Kinase; D009218:Myosins