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J. Biol. Chem., Vol. 280, Issue 24, 22769-22775, June 17, 2005

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

Kye-Young Kim¶, Mihály Kovács||, Sachiyo Kawamoto, James R. Sellers||, and Robert S. Adelstein**

From the Laboratory of Molecular Cardiology and ^||Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

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.

Received for publication, March 30, 2005

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to the memory of Yvette A. Preston.

These authors made major contributions to this work.

^¶ Partially supported by the Korea Research Foundation Grant funded by Korea Government (Ministry of Education and Human Resources Development, Basic Research Promotion Fund M01–2003-000-10307-0).

^** To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 8N202, 10 Center Dr. MSC 1762, Bethesda, MD. 20892-1762; Tel.: 301-496-1865; Fax: 301-402-1542; E-mail: AdelsteR{at}NHLBI.NIH.gov.

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