Chimeric Substitutions of the Actin-binding Loop Activate Dephosphorylated but Not Phosphorylated Smooth Muscle Heavy Meromyosin

doi:10.1074/jbc.270.51.30260

Chimeric Substitutions of the Actin-binding Loop Activate Dephosphorylated but Not Phosphorylated Smooth Muscle Heavy Meromyosin (*)

From the (1)Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405 and
(2)Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110

^§Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 617-736-2448; Fax: 617-736-2405; trybus{at}hydra.rose.brandeis.edu.

Abstract

Regulatory light chain (RLC) phosphorylation is necessary to activate smooth muscle myosin, unlike constitutively active striated muscle myosins. Here we show that an actin-binding surface loop located at the 50/20-kDa junction contributes to this fundamental difference between myosins. Substitution of the native actin-binding loop of smooth muscle heavy meromyosin (HMM) with that from either skeletal or β-cardiac myosin caused the chimeric HMMs to become unregulated like the myosin from which the loop was derived. Dephosphorylated chimeric HMMs gained the ability to move actin in a motility assay and had 50-70% of the actin-activated ATPase activity of phosphorylated wild-type HMM. Direct binding measurements showed that the affinity of HMM for actin in the presence of MgATP was unaffected by loop substitution; thus the rate of a step other than binding is increased. Phosphorylation of the chimeras did not lead to a higher V_max than obtained for wild-type HMM. In the absence of actin, a foreign loop did not affect nucleotide trapping. Native regulated molecules have thus evolved a loop sequence which prevents rapid product release by actin when the RLC is dephosphorylated, thereby allowing activity to be controlled by RLC phosphorylation.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL38113 (to K. M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HMM

heavy meromyosin

ABL

actin-binding loop at the 50/20-kDa interface of the myosin heavy chain

CABL

β-cardiac actin-binding loop

SABL

skeletal actin-binding loop

HC

myosin heavy chain

PCR

polymerase chain reaction

RLC

regulatory light chain

WT

wild-type.
- Received September 25, 1995.
- Revision received October 23, 1995.