Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C.
Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.
Kim
Kye-Young
KY
Laboratory of Molecular Cardiology and Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kovács
Mihály
M
Kawamoto
Sachiyo
S
Sellers
James R
JR
Adelstein
Robert S
RS
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D017398:Alternative Splicing; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D015879:Myosin Subfragments / Q000737:chemistry; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics*; D009218:Myosins / Q000737:chemistry; D024501:Nonmuscle Myosin Type IIB / Q000737:chemistry* / Q000235:genetics*; D010641:Phenotype; D017354:Point Mutation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D000199:Actins; D000244:Adenosine Diphosphate; D001120:Arginine; D001216:Asparagine; D017301:Ca(2+) Mg(2+)-ATPase; D018995:Myosin Heavy Chains; D015879:Myosin Subfragments; D024462:Myosin Type II; D009218:Myosins; D024501:Nonmuscle Myosin Type IIB; D020033:Protein Isoforms; D011994:Recombinant Proteins; C477830:MYH7b protein, human; C483905:Myh14 protein, mouse