Recently, an isoform of vertebrate nonmuscle myosin heavy chain-B (MHC-B) with an inserted sequence of 10 amino acids and isoforms of smooth muscle MHC-204 and MHC-200, with an insertion of 7 amino acids 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. In nonmuscle tissue, the insert contains a consensus sequence for phosphorylation by cyclin-p34cdc2 (cdc2) kinase, a cell-cycle regulated kinase that catalyzes entry of cells into meiosis and mitosis. Using myosin immunoprecipitated from cultured Xenopus XTC cells, we found that cdc2 kinase catalyzes the phosphorylation of the inserted, but not the noninserted, form of the MHC in vitro. One- dimensional tryptic phosphopeptide maps of MHC-B phosphorylated in vitro with cdc2 kinase revealed one major phosphopeptide. This phosphopeptide was purified by reverse-phase HPLC and sequenced. The sequence obtained was DHTIPTESPK. Phosphoamino acid analysis demonstrated that the serine was the phosphorylated residue. This serine is followed by a proline and a basic residue which is typical of cdc2 kinase phosphorylation sites. By comparison with the deduced amino acid sequence for Xenopus MHC-B, we have localized the phosphorylated serine to residue 214 which is within the insert near the ATP-binding region. Preliminary results using intact XTC cells show a higher level of phosphorylation of the inserted, but not the noninserted, MHC in mitotic compared to interphase cells. Tryptic phosphopeptide maps of MHC-B phosphorylated in intact cells during log phase of growth show a phosphopeptide which co-migrates with the tryptic phosphopeptide obtained from MHC-B phosphorylated in vitro with cdc2 kinase. These results suggest that Ser 214 may be phosphorylated in vivo by cdc2 kinase or a cdc2-like kinase.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL004210-06
Application #
3779594
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
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