2 PHOSPHORYLATIONS SPECIFIC TO THE TAIL REGION OF THE 204-KDA HEAVY-CHAIN ISOFORM OF PORCINE AORTA SMOOTH-MUSCLE MYOSIN

In a porcine aorta extract, we observed two protein kinase activities which specifically phosphorylate the 204-kDa heavy chain isoform of ao rta myosin in the absence of conventional kinase activators, We referr ed to these two protein kinases, eluted at 0.15 and 0.2 M KCl from a D EAE-column, as myosin kinases I (MKI) and II (MKII), respectively. The phosphorylation site for MKI was determined using a purified phosphop eptide derived from porcine aorta myosin phosphorylated with MKI. By c omparison with the deduced amino acid sequence for smooth muscle myosi ns, the site corresponded to a Ser located at 3 amino acids upstream f rom a Pro, the putative end of the alpha-helical segment of the 204-kD a 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 w as purified to near homogeneity. MKII phosphorylated the porcine aorta myosin heavy chain at a Ser 19 amino acids downstream from the MKI si te. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site for casein kinase II and was homologous t o 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.