title: 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.
abstract: 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 MIIB alpha and MIIB beta, 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. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta 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 MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha 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 MIIB beta 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 MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha 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 MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) 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.
authors.affiliation: Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. PJISADOG@interport.com
authors.forename: N
authors.initials: N
authors.lastname: Murakami
authors.forename: V P
authors.initials: VP
authors.lastname: Chauhan
authors.forename: M
authors.initials: M
authors.lastname: Elzinga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D047390:Casein Kinase II; D002417:Cattle; D002645:Chickens; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013552:Swine
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D018995:Myosin Heavy Chains