PHOSPHOLIPID-BINDING, PHOSPHORYLATION BY PROTEIN-KINASE-C, AND FILAMENT ASSEMBLY OF THE COOH TERMINAL HEAVY-CHAIN FRAGMENTS OF NONMUSCLE MYOSIN-II ISOFORMS MIIA AND MIIB

Previously, we showed that myosin II heavy chains bind to phosphatidyl serine (PS) liposomes via their COOH terminal regions and that protein kinase C (PK C) phosphorylates the PS-bound heavy chains [Murakami et al. (1994) J. Biol. Chem. 269, 16082-16090]. In this report, we studi ed the phospholipid binding, the kinetics of phosphorylation by PK C, and the effect of PK C-mediated phosphorylation on assembly using 46-4 7 kDa fragments from the COOH termini of macrophage (MIIA(F46)) and br ain type (MIIB(F47)) heavy chain isoforms. Binding of the fragments to PS or phosphatidylinositol liposomes increased turbidity, but MIIA(F4 6) gave higher turbidity than MIIB(F47) Both fragments were sedimented similarly by ultracentrifugation in PS concentration and mole percent of PS dependent manners. With mixed PS/phosphatidylcholine (PC) lipos omes, at least 70 mol % PS was required for heavy chain binding. A sim ilar level of PS was required for phosphorylation of fragments by PK C , indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PK C. PK C phosphorylated MIIB(F47) with V-max valu es 4-5 times higher than those of MIIA(F46), but the K-m values for th e two substrates were similar. The apparent K-m values for PS liposome s (K-lipid) were also similar for phosphorylation of both isoforms. Mi xing PS with PC increased the K-lipid and reduced the V-max values but did not alter the K-m values for the substrates. Assembly of MIIB(F47 ), but not MIIA(F46), was significantly inhibited by the phosphorylati on, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PK C.