The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I. An improved method for locating the phosphorylated amino acid.
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.
Brzeska
H
H
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Lynch
T J
TJ
Martin
B
B
Korn
E D
ED
D000048:Acanthamoeba / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D005796:Genes; D007527:Isoenzymes / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin
D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins