Disease-associated Mutations and Alternative Splicing Alter the Enzymatic and Motile Activity of Nonmuscle Myosins II-B and II-C

• Published in: The Journal of biological chemistry Vol. 280; no. 24; pp. 22769 - 22775
• Main Authors: Kim, Kye-Young, Kovács, Mihály, Kawamoto, Sachiyo, Sellers, James R, Adelstein, Robert S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 17.06.2005
• Subjects: 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; Insecta; 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
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M503488200

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.