Protein Kinase C Phosphorylates Nonmuscle Myosin-II Heavy Chain from Drosophila but Regulation of Myosin Function by This Enzyme Is Not Required for Viability in Flies

摘要

Conventional myosins (myosin-IIs) generate forceS for cell shape change and cell motility. fyosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be nportant in metazoans. To investigate this regulation in a complex eukaryote, we purified the Drosophila lyosin-II tail expresse~ in Escherichia coli and showed that it was phosphorylated in vitro by protein inase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These tes are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosiri-IIs. If the !/0 serines are mutagenized to alanine or. aspartic acid, phosphorylation no longer occurs. Using a 341 mino acid tail fragment, we show that. there is no difference in the si1lt-dependent assembly of wild-type hosphotylatedand mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, 'hich is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, we enerated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or ear-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue le lethal phenotype due to severe zipper mutations. These results suggest that while the myosiri-II heavy bain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. ~onservation during 530-1000 million years of evolution suggests that regulation by heavy chain hosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way.