Two 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 phosphorylation sites

摘要

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIBalpha and MIIBbeta, in rabbit 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 form in rabbit brain, while the B-beta form was novel. MIIBbeta F47 differed from MIIBalpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain, in MIIBbeta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIBalpha F47 and MIIBbeta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIBbeta F47 and the nonhelical domain of MIIBalpha F47 behaved 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 phosphate/mol of MIIBalpha F47 and MIIBbeta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIBalpha F47 but only slight inhibition for MIIBbeta F47. PK C sites in MIIBalpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites 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 sites were identified within the COOH end of MIIBalpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4)-E . The same sites were phosphorylated 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 were phosphorylated in MIIBbeta F47, while in MIIBalpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (I) the helical domains dictate the intrinsic salt dependence of assembly 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 phosphorylation on assembly are isoform specific. [References: 45]