A Xenopus Nonmuscle Myosin Heavy Chain Isoform Is Phosphorylated by Cyclin-p34 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. Recent work has identified additional, alternatively spliced isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides (chicken and human) or 48 nucleotides ( Xenopus ) at a site corresponding to the ATP binding region in the MHC protein (Takahashi, M., Kawamoto, S., and Adelstein, 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 contains a consensus sequence for phosphorylation by cyclin-p34 (cdc2) kinase. In cultured Xenopus XTC cells, we have identified two inserted MHC-B isoforms and a noninserted MHC-A isoform by immunoblotting 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 MHC-B isoforms but not MHC-A. Isoelectric 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 site within the insert near the ATP binding region. The same site was phosphorylated in intact XTC cells during log phase of growth and in cell-free lysates of Xenopus eggs stabilized in second meiotic metaphase but not interphase. Moreover, Ser-214 phosphorylation was detected during maturation of Xenopus oocytes when the cdc2 kinase-containing maturation-promoting factor was activated, but not in G 2 interphase-arrested oocytes. These results demonstrate that MHC-B phosphorylation is tightly regulated by cdc2 kinase during meiotic cell cycles. Furthermore, MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve different functions in these cells.