2 NONMUSCLE MYOSIN-II HEAVY-CHAIN ISOFORMS EXPRESSED IN RABBIT BRAINS- FILAMENT FORMING PROPERTIES, THE EFFECTS OF PHOSPHORYLATION BY PROTEIN-KINASE-C AND CASEIN KINASE-II, AND LOCATION OF THE PHOSPHORYLATIONSITES

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two cl osely related forms of MIIB, designated MIIBalpha and MIIBbeta, in rab bit brains. The B-alpha form corresponded to SMemb, described by Kuro- o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant f orm in rabbit brain, while the B-beta form was novel. MIIBbeta F47 dif fered from MIIBalpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain, in MIIBbeta F47, Ser, Pro, an d Lys replaced Pro, Ser, and Glu, respectively. MIIBalpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of v arious concentrations of salt, and a chimera containing the helical do main of MIIBbeta F47 and the nonhelical domain of MIIBalpha F47 behave d very much like MIIBbeta F47, Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIBalpha F47 and MIIBbeta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms, Casein kinase II (CK II) incorporated 4 and 2 mol of ph osphate/mol of MIIBalpha F47 and MIIBbeta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIBalpha F47 but on ly slight inhibition for MIIBbeta F47. PK C sites in MIIBalpha F47 wer e localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO 4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C si tes, only one site seemed to be phosphorylated per peptide. The PK C s ites in MIIBbeta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4) -, with total incorporation of about 2 mol/mol of peptide. In addition , PK C phosphorylated a Ser within the predicted helical domain, E-V-S (PO4)-T-L, in both MIIBalpha F47 and MIIBbeta F47. For CK II, five sit es were identified within the COOH end of MIIBalpha F47: -T(PO4)-E-S-K -T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4)-E. The same sites were phosphorylat ed in MIIBbeta F47 except for the first Ser, which was replaced by Pro in MIIBbeta F47 An average of about two of the four potential sites w ere phosphorylated in MIIBbeta F47, while in MIIBalpha F47 all five si tes could be fully phosphorylated by CK II. Our results demonstrate th at (I) the helical domains dictate the intrinsic salt dependence of as sembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH -terminal nonhelical domain, and (3) the effects of the phosphorylatio n on assembly are isoform specific.