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J Biol Chem. 1989 Nov 15;264(32):19340-8.

The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I. An improved method for locating the phosphorylated amino acid.

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  • 1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.

Abstract

The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB, and IC are expressed only when a single site in their heavy chains is phosphorylated by a myosin I heavy chain-specific kinase. We show that phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in myosin IC, and a threonine residue at a corresponding position in myosin IA whose amino acid sequence is as yet unknown. The most obvious feature common to the three substrates is a basic amino acid(s) 2 or 3 residues before the site of phosphorylation. The phosphorylation site is located between the ATP- and actin-binding sites, which corresponds to the middle of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence similarity between the region surrounding the phosphorylation site of myosin I and subfragment 1 is much lower than the average sequence similarity between myosin I and subfragment 1. This is consistent with the hypothesis that the conformation of this region of myosin I differs from that of the corresponding region in skeletal muscle myosin and that phosphorylation converts the conformation of the actomyosin I complex into a conformation comparable to that present in actosubfragment 1 without phosphorylation. The protein sequences obtained in the course of this work led to the conclusion that the myosin I genes previously identified as myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and IB heavy chains, respectively. Finally, we report a modification of the method for monitoring the appearance of 32Pi during sequencing of 32P-labeled peptides that results in almost complete recovery of the radioactivity, thus allowing unequivocal assignment of the position of the phosphorylated residue.

PMID:
2530230
[PubMed - indexed for MEDLINE]
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