Recently, an isoform of vertebrate nonmuscle myosin heavy chain B (MHC-B) with an inserted sequence of 10 or 16 amino acids (Takahashi et al., J. Biol. Chem. 267: 17864, 1992; Bhatia-Dey et al., Proc. Natl. Acad. Sci. USA 90: 2856, 1993) and isoforms of smooth muscle MHC-204 and MHC-200, with an insertion of 7 amino acids (Kelley et al., J. Biol. Chem. 268: 12848, 1993) at a site near the ATP binding region in the myosin head, have been reported. In smooth muscle, the insert occurs in intestinal, but not vascular, myosin. The presence of the insert correlates with a faster velocity of movement of actin filaments in an in vitro motility assay and a higher actin-activated Mg2+-ATPase activity. Preliminary evidence suggests that the insert increases the rate of ADP release, thereby increasing enzymatic activity. In nonmuscle MHC-B, the deduced amino acid sequence of the insert 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 non-inserted 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 the inserted MHC-B isoforms only. Isoelectric focusing of tryptic peptides generated from MHC-B phosphorylated with cdc2 kinase revealed one major phosphopeptide which was purified by reverse-phase HPLC 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 extracts 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 is activated, but not in G2 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.
