Chimeric substitutions of the actin-binding loop activate dephosphorylated but not phosphorylated smooth muscle heavy meromyosin.
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 beta-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 Vmax 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.
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington 05405, USA.
A S
AS
Rovner
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Y
Freyzon
K M
KM
Trybus
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002645:Chickens; D005895:Gizzard, Avian; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000378:metabolism*; D018411:Spodoptera; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014422:Turkeys
D000199:Actins; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D009218:Myosins