Phospholipid binding, phosphorylation by protein kinase C, and filament assembly of the COOH terminal heavy chain fragments of nonmuscle myosin II isoforms MIIA and MIIB - PubMed

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• PMID: 8519761
• DOI: 10.1021/bi00049a019

Comparative Study

Phospholipid binding, phosphorylation by protein kinase C, and filament assembly of the COOH terminal heavy chain fragments of nonmuscle myosin II isoforms MIIA and MIIB

N Murakami et al. Biochemistry. 1995.

Abstract

Previously, we showed that myosin II heavy chains bind to phosphatidylserine (PS) liposomes via their COOH terminal regions and that protein kinase C (PK C) phosphorylates the PS-bound heavy chains [Murakami et al. (1994) J. Biol. Chem. 269, 16082-16090]. In this report, we studied the phospholipid binding, the kinetics of phosphorylation by PK C, and the effect of PK C-mediated phosphorylation on assembly using 46-47 kDa fragments from the COOH termini of macrophage (MIIAF46) and brain type (MIIBF47) heavy chain isoforms. Binding of the fragments to PS or phosphatidylinositol liposomes increased turbidity, but MIIAF46 gave higher turbidity than MIIBF47. Both fragments were sedimented similarly by ultracentrifugation in PS concentration and mole percent of PS dependent manners. With mixed PS/phosphatidylcholine (PC) liposomes, at least 70 mol % PS was required for heavy chain binding. A similar level of PS was required for phosphorylation of fragments by PK C, indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PK C. PK C phosphorylated MIIBF47 with Vmax values 4-5 times higher than those of MIIAF46, but the Km values for the two substrates were similar. The apparent Km values for PS liposomes (Klipid) were also similar for phosphorylation of both isoforms. Mixing PS with PC increased the Klipid and reduced the Vmax values but did not alter the Km values for the substrates. Assembly of MIIBF47, but not MIIAF46, was significantly inhibited by the phosphorylation, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PK C.

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