Abstract
Acanthamoeba myosin I heavy chain kinase activates the actin-activated Mg2+ -ATPase activity of the Acanthamoeba myosin I isoenzymes, myosins IA and IB, by phosphorylating a single site within the myosin heavy chain. In this paper, we report that myosin I heavy chain kinase also phosphorylates isolated turkey gizzard smooth muscle myosin light chains, gizzard smooth muscle heavy meromyosin, and intact gizzard smooth muscle myosin, all in the absence of Ca2+ and with specific activities close to those measured for purified Ca2+/calmodulin-dependent gizzard smooth muscle myosin light chain kinase. Myosin I heavy chain kinase incorporates a maximum of 2 mol of phosphate/mol of heavy meromyosin, both by itself and together with smooth muscle myosin light chain kinase (the light chain kinase alone incorporates 1.6 mol of phosphate/mol of heavy meromyosin). Both kinases phosphorylate intact smooth muscle myosin to a maximum of 2 mol of phosphate/mol of myosin. Myosin I heavy chain kinase fully activates the actin-activated Mg2+ -ATPase of both myosin and heavy meromyosin. Two-dimensional tryptic peptide maps of isolated light chains phosphorylated by myosin I kinase show the same phosphopeptide as for light chains phosphorylated by the light chain kinase. These results support the conclusion that myosin I heavy chain kinase phosphorylates gizzard smooth muscle myosin at the same site within the 20,000-Da light chain as does smooth muscle myosin light chain kinase. The results suggest that the amino acid sequence around the phosphorylation site within the heavy chain of Acanthamoeba myosin I isoenzymes may be similar to the primary sequence around the phosphorylation site within the smooth muscle myosin light chain.
Highlights
THEJOURNALOF BIOLOGICACHLEMISTRYPhosphorylation and Activation of Smooth Muscle Myosinby Acanthamoeba Myosin I Heavy Chain Kinase*
IB, by phosphorylating a single site within themyosin that are well below saturating levels [1].The kinase requires heavy chain
We extend this observation, showing that myosinI heavy chainkinasephosphorylates intact gizzard smooth muscle myosin and heavy meromyosin at high rates, thatmyosin I heavychain kinasefully activates with smoothmuscle myosin light chain kinase
Summary
Phosphorylation and Activation of Smooth Muscle Myosinby Acanthamoeba Myosin I Heavy Chain Kinase*. Phosphate-free turkeygizzard total smooth muscle in the case of myosin IB [1].Maximal phosphorylation of light chains (6:4 ratio, 20,000- to 17,000-Da lightchain) were prepared myosin IA and IB resultsin a 20-fold increase in their actin- from purified myosin as described by Sellers et al [10]. The rate was obtained from the linear portion of the time course (usually from time 0 to 3 min) Both myosin I kinase and smooth muscle light chain were devoid of M%f-ATPase activity. Heavy meromyosin, 30 mM Tris (pH 7.41, 4 mM magnesium acetate, Peptide Mapping-Two-dimensional cellulose thin layer maps of 0.2 mM CaCI2,0.25 mM dithiothreitol, and 2.0 mM [-y-32P]ATP(75 tryptic peptides of phosphorylated smoothmuscle myosin light chains pCi/wmol) in a final volume of 55 p1.
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