Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii.
In previous work from this laboratory, a partially purified protein kinase from the soil amoeba Acanthamoeba castellanii was shown to phosphorylate the heavy chain of the two single-headed Acanthamoeba myosin isoenzymes, myosin IA and IB, resulting in a 10- to 20-fold increase in their actin-activated Mg2+-ATPase activities (Maruta, H., and Korn, E.D. (1977) J. Biol. Chem. 252, 8329-8332). A myosin I heavy chain kinase has now been purified to near homogeneity from Acanthamoeba by chromatography on DE-52 cellulose, phosphocellulose, and Procion red dye, followed by chromatography on histone-Sepharose. Myosin I heavy chain kinase contains a single polypeptide of 107,000 Da by electrophoretic analysis. Molecular sieve chromatography yields a Stokes radius of 4.1 nm, consistent with a molecular weight of 107,000 for a native protein with a frictional ratio of approximately 1.3:1. The kinase catalyzes the incorporation of 0.9 to 1.0 mol of phosphate into the heavy chain of both myosins IA and IB. Phosphoserine has been shown to be the phosphorylated amino acid in myosin IB. The kinase has highest specific activity toward myosin IA and IB, about 3-4 mumol of phosphate incorporated/min/mg (30 degrees C) at concentrations of myosin I that are well below saturating levels. The kinase also phosphorylates histone 2A, isolated smooth muscle light chains, and, to a very small extent, casein, but has no activity toward phosvitin or myosin II, a third Acanthamoeba myosin isoenzyme with a very different structure from myosin IA and IB. Myosin I heavy chain kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or cAMP for activity. The kinase undergoes an apparent autophosphorylation.
D000656:Amoeba / Q000201:enzymology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D011189:Potassium Chloride / Q000494:pharmacology; D015800:Protozoan Proteins
D006657:Histones; D010710:Phosphates; D015800:Protozoan Proteins; D010768:Phosphoserine; D011189:Potassium Chloride; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D008274:Magnesium