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.

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

“…In favor of this view, Burridge and coworkers suggested that the activation of myosin II activity produces the force driving the formation of stress fibers and focal adhesions (Chrzanowska-Wodnicka and Burridge, 1996). Because stress fibers are thought to attach to the focal adhesions that provide the force required for anchoring them to the cell matrix , it is therefore likely that the decrease in the myosin II-driven force by the phosphorylation of MLC 20 at the inhibitory sites influences the formation of focal adhesions as well as the stress fibers.Previous in vitro biochemical studies have suggested that the phosphorylation of myosin II at the nonhelical tail plays a role in the disassembly of myosin filamentous structures (Murakami et al, 1998(Murakami et al, , 2000Dulyaninova et al, 2005). In the present study, the expression of S1A/S2A MLC 20 attenuated the PDGF-induced disassembly of stress fibers (Figure 5), whereas the expression of ⌬C-Myosin IIB did not significantly change the disassembly of stress fibers (Figure 6).…”

“…Moreover, it was recently reported that myosin II isoforms (IIA and IIB) differentially contributes to the cell motility (Even-Ram et al, 2007;Vicente-Manzanares et al, 2007), suggesting that myosin isoforms are regulated by distinct mechanisms in motile cells. Supporting this notion, biochemical studies revealed that phosphate incorporation of MHC-IIB by PKC is much faster than that of myosin IIA and, in contrast, that filament assembly of myosin IIA is regulated by its binding protein (Mts1) but not that of myosin IIB (Murakami et al, 1998(Murakami et al, , 2000Dulyaninova et al, 2005). To clarify the roles of phosphorylation of MLC 20 and MHC on actomyosin dynamics in distinct cellular domains will be the subjects of future work.…”

“…Previous studies have reported that PKC phosphorylates the heavy chains of nonmuscle myosin IIB in the nonhelical tailpiece, resulting in the inhibition of the assembly of myosin IIB into filaments in vitro (Murakami et al, 1998;Bresnick, 1999). Although it is not known if PDGF stimulation induces the phosphorylation of myosin IIB heavy chain, we examined whether the heavy-chain phosphorylation is involved in the regulation of the disassembly of stress fibers in cells.…”

“…Although it is not known if PDGF stimulation induces the phosphorylation of myosin IIB heavy chain, we examined whether the heavy-chain phosphorylation is involved in the regulation of the disassembly of stress fibers in cells. NIH3T3 cells were transfected with yellow fluorescent protein (YFP)-tagged ⌬C-Myosin IIB in which the nonhelical tail sequence containing multiple phosphorylation sites is deleted (Murakami et al, 1990(Murakami et al, , 1998. The expression level of wild-type and ⌬C-Myosin IIB in cells was estimated by measuring the fluorescence intensity of YFP signals, and the cells that expressed a similar level of the wild-type or ⌬C-Myoisn IIB were used for the experiment.…”

Conventional myosins – unconventional functions

“…Exactly how these observations fit alongside the expected decline in activity following specific Ser1937 phosphorylation by PKCζ (Even-Faitelson and Ravid 2006) remains unclear, but the observed differences presumably relate to the consequences of phosphorylation at additional sites by PKCγ (Rosenberg and Ravid 2006). PKCβII (Table 1) phosphorylates the HCs of both M2A and M2B; the former on Ser1917, a site close to the nonhelical tailpiece but still within the helical domain (Ludowyke et al 2006), and the latter at multiple sites within the nonhelical tailpiece (Murakami et al 1998). PKCβII-dependent phosphorylation of the M2A HC in RBL-2H3 Mast cells occurs as a consequence of antigen binding to IgE and the phosphorylation time-course parallels degranulation (Ludowyke et al 2006).…”