The ATPase activity of Myr3, a rat myosin I, is allosterically inhibited by its own tail domain and by Ca2+ binding to its light chain calmodulin.
We purified Myr3 (third unconventional myosin from rat), a mammalian "amoeboid" subclass myosin I, from rat liver. The heavy chain of purified Myr3 is associated with a single calmodulin light chain. Myr3 exhibits K/EDTA-ATPase and Mg-ATPase activity. The Mg-ATPase activity is stimulated by increasing F-actin concentrations in a complex triphasic manner similar to the Mg-ATPase activity of myosin I molecules from protozoa. Although purified Myr3 was observed to cross-link actin filaments, it bound in an ATP regulated manner to F-actin, and no evidence for a nucleotide-independent high affinity actin binding site that could explain the triphasic activation pattern was obtained. Micromolar concentrations of free Ca2+ reversibly inhibit the Mg-ATPase activity of Myr3 by binding to its light chain calmodulin, which remains bound to the Myr3 heavy chain irrespective of the free Ca2+ concentration. Polyclonal antibodies and Fab fragments directed against the tail domain were found to stimulate the Mg-ATPase activity. A similar stimulation of the Myr3 Mg-ATPase activity is observed upon proteolytic removal of the very C-terminal SH3 domain. These results demonstrate that Myr3 is subject to negative regulation by free calcium and its own tail domain and possibly positive regulation by a tail-domain binding partner.
Stöffler
H E
HE
Adolf-Butenandt-Institut, Zellbiologie, Ludwig-Maximilians-Universität, D-80336 München, Germany.
Bähler
M
M
D000199:Actins / Q000378:metabolism / Q000648:ultrastructure; D000494:Allosteric Regulation / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D008099:Liver / Q000737:chemistry; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism; D024461:Myosin Type I; D009218:Myosins / Q000737:chemistry* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D018909:src Homology Domains / Q000502:physiology
D000199:Actins; D000906:Antibodies; D002147:Calmodulin; D007140:Immunoglobulin Fab Fragments; C503808:Myo1e protein, rat; D018994:Myosin Light Chains; D010446:Peptide Fragments; D017301:Ca(2+) Mg(2+)-ATPase; D024461:Myosin Type I; D009218:Myosins; D002118:Calcium