PMID:15845534

Citation	Kim, KY, Kovács, M, Kawamoto, S, Sellers, JR and Adelstein, RS (2005) Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C. J. Biol. Chem. 280:22769-75
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M503488200
Keywords	Actins/chemistry; Adenosine Diphosphate/chemistry; Alternative Splicing; Animals; Arginine/chemistry; Asparagine/chemistry; Ca(2+) Mg(2+)-ATPase/chemistry; Dose-Response Relationship, Drug; Genetic Vectors; Humans; Insects; Kinetics; Mice; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Myosin Heavy Chains/chemistry; Myosin Heavy Chains/genetics; Myosin Subfragments/chemistry; Myosin Type II/chemistry; Myosin Type II/genetics; Myosins/chemistry; Nonmuscle Myosin Type IIB/chemistry; Nonmuscle Myosin Type IIB/genetics; Phenotype; Point Mutation; Protein Isoforms; Protein Structure, Tertiary; Recombinant Proteins/chemistry
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