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^†

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIB^F47), we found two closely related forms of MIIB, designated MIIB^α and MIIB^β, in rabbit brains. The B^α form corresponded to SM_emb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B^β form was novel. MIIB^βF47 differed from MIIB^αF47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB^βF47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB^αF47 and MIIB^βF47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB^βF47 and the nonhelical domain of MIIB^αF47 behaved very much like MIIB^βF47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB^αF47 and MIIB^βF47, 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^αF47 and MIIB^βF47 peptides, respectively, and this caused strong inhibition of assembly for MIIB^αF47 but only slight inhibition for MIIB^βF47. PK C sites in MIIB^αF47 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^βF47 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^αF47 and MIIB^βF47. For CK II, five sites were identified within the COOH end of MIIB^αF47:  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^βF47 except for the first Ser, which was replaced by Pro in MIIB^βF47. An average of about two of the four potential sites were phosphorylated in MIIB^βF47, while in MIIB^αF47 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.