A XENOPUS NONMUSCLE MYOSIN HEAVY-CHAIN ISOFORM IS PHOSPHORYLATED BY CYCLIN-P34(CDC2) KINASE DURING MEIOSIS

There are two vertebrate nonmuscle myosin heavy chain (MHC) genes that encode two separate isoforms of the heavy chain, MHC-A and MHC-B. Rec ent work has identified additional, alternatively spliced isoforms of MRC-R cDNA with inserted sequences of 30 nucleotides (chicken and huma n) or 48 nucleotides (Xenopus) at a site corresponding to the ATP bind ing region in the MHC protein (Takahashi, M., Kawamoto, S. and Adelste in, R. S. (1992) J. Biol. Chem. 267, 17864-17871) and Bhatia-Dey, N., Adelstein, R. S., and Dawid, I. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2856-2859). The deduced amino acid sequence of these inserts c ontains a consensus sequence for phosphorylation by cyclin-p34(cdc2) ( cdc2) kinase. In cultured Xenopus XTC cells, we have identified two in serted MHC-B isoforms and a noninserted MHC-A isoform by immunoblottin g of cell extracts. When myosin was immunoprecipitated from XTC cells and phosphorylated in vitro with cdc2 kinase, the kinase catalyzed the phosphorylation of both inserted MRC-B isoforms but not MHC-A. Isoele ctric focusing of tryptic peptides generated from MHC-B phosphorylated with cdc2 kinase revealed one major phosphopeptide that was purified by reverse phase high performance liquid chromatography and sequenced. The phosphorylated residue was Ser-214, the cdc2 kinase consensus sit e within the insert near the ATP binding region. The same site was pho sphorylated in intact XTC cells during log phase of growth and in cell -free lysates of Xenopus eggs stabilized in second meiotic metaphase b ut not interphase. Moreover, Ser-214 phosphorylation was detected duri ng maturation of Xenopus oocytes when the cdc2 kinase-containing matur ation-promoting factor was activated, but not in G(2) interphase-arres ted oocytes. These results demonstrate that MBC-B phosphorylation is t ightly regulated by cdc2 kinase during meiotic cell cycles. Furthermor e, MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve different functions in these c ells.