Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells.

L B Ray,T W Sturgill

doi:10.1016/s0021-9258(18)37813-x

Abstract

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.

Highlights

ChromatographicProperties of Insulin-stimulated MAP Kinase-Extract supernatants of 3T3-Ll cells which have been treated with insulin (80 nM) for 10 min contain MAP kinase activity that is stimulated 1.5-3-foldabove that in extracts of control cells (17)
The increased activity persisted after chromatography of such extracts on DEAE-cellulose (Fig. 1).The insulin-stimulated MAP kinase activity was retained on the column and eluted by a linear gradientof NaC1
We have identified a soluble protein kinase from 3T3-Ll cells which phosphorylates microtubule-associated protein 2 from bovine brain in vitro (16, 17)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Preparation of Extract Supernatants”BT3-Ll cells were grown and differentiated by the methods of Rubin et al (20) and used on days 5-8. Assays to determine substrate specificity of the purified fraction were performed at 30 “C for 15 min in a 60-pl reaction volume containing 5 pl of the enzyme fraction, 50 mM @-glycerolphosphate, pH 7.5, 1 mM dithiothreitol, 10 mM magnesium acetate, 40 p M [T-~’P]ATP(1 cpm/fmol), and either 0.1 mg/ml MAP-2 in the presence of 1mg/ml BSA as carrier or 0.5 mg/ml histone IIA or 111s (Sigma), histone HA2B or HF2B (Worthington),dephosphocasein (Sigma), phosvitin (Sigma), protamine sulfate (Lilly), or 40 S ribosomal subunits from Artemia salina (17). Storage of Active Enzyme-Peak fractions from phenyl-Superose chromatography were combined and BSA (Pentex, crystallized, Miles Laboratories Inc.) added to yield 0.3 mg of protein/ml This sample was applied to a Centricon microconcentrator (Amicon), centrifuged as recommended by the manufacturer for 1.5-2 h, collected, and stored at -70 “C. Materials-Bovine insulin and protamine sulfate were gifts from Lilly. [Y-~’P]ATPwas synthesized by the method of Johnson and Walseth (24); [Y-~’P]GTPwas from Du Pont-New England Nuclear

RESULTS

40 S ribosomal subunits

DISCUSSION

Methods