Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells.
A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.
Ray
L B
LB
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville 22908.
Sturgill
T W
TW
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine / Q000031:analogs & derivatives*; D013379:Substrate Specificity; D013912:Threonine / Q000031:analogs & derivatives*
D007328:Insulin; D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases