In a porcine aorta extract, we observed two protein kinase activities which specifically phosphorylate the 204-kDa heavy chain isoform of aorta myosin in the absence of conventional kinase activators. We referred to these two protein kinases, eluted at 0.15 and 0.2 M KCl from a DEAE-column, as myosin kinases I (MKI) and II (MKII), respectively. The phosphorylation site for MKI was determined using a purified phosphopeptide derived from porcine aorta myosin phosphorylated with MKI. By comparison with the deduced amino acid sequence for smooth muscle myosins, the site corresponded to a Ser located at 3 amino acids upstream from a Pro, the putative end of the alpha-helical segment of the 204-kDa heavy chain tail. A homologous Ser is only present in smooth muscle myosins, i.e. not in nonmuscle myosins. MKI was purified 130-fold, but not separated from a kinase activity phosphorylating Ser1 or Ser2 in the 20-kDa regulatory light chain of aorta myosin. In contrast, MKII was purified to near homogeneity. MKII phosphorylated the porcine aorta myosin heavy chain at a Ser 19 amino acids downstream from the MKI site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site for casein kinase II and was homologous to that reported for bovine aorta myosin [Kelley, C.A. and Adelstein, R.S. (1990) J Biol. Chem. 265, 17876-17882]. MKII was identified as a multifunctional protein kinase, casein kinase II.
As reported previously, the synthetic heptapeptide having the amino acid sequence around the reactive Cys (SH1) of myosin heavy chain, IRICRKG-NH2, inhibited acto-myosin subfragment-1 (S-1) ATPase activity and half inhibition (K1/2) was observed at a peptide concentration of 0.06 mM. The inhibitory ability of the peptide was found to be decreased to one-fifth by acetylation of its N-terminal alpha-amino group. A similar effect of N-acetylation was observed with a nonapeptide, EGIRICRKG-NH2, and an undecapeptide, VLEGIRICRKG-NH2. These results indicate that N-terminal-free synthetic peptides do not act as proper analogs of the corresponding segment of S-1 heavy chain against F-actin. We isolated a longer peptide extending from Thr682 to Lys709 in S-1 heavy chain, with two Cys residues corresponding to SH1 and SH2. This peptide, having 28 residues (28peptide), inhibited acto-S-1 ATPase activity with a K1/2 of 0.23 mM. A cosedimentation binding assay indicated that the 28peptide completely dissociated acto-S-1 in the presence of ATP. This behavior is different from that observed with the N-terminal-free synthetic heptapeptide, and thus the 28peptide might be an analog of the corresponding segment. There is a possibility that the region corresponding to the 28peptide in S-1 heavy chain may bind directly with F-actin and may be involved in determining the acto-S-1 link during the steady state of the acto-S-1 ATPase reaction.
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