Identification and Characterization of a Mitogen-Activated S6 Kinase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 2; pp. 406 - 410
• Main Authors: Jenö, Paul, Ballou, Lisa M., Novak-Hofer, Ilse, Thomas, George
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.01.1988; National Acad Sciences
• Subjects: 3T3 cells; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; cell culture; Cell extracts; Cells, Cultured; Enzyme Activation; Enzymes; Enzymes and enzyme inhibitors; Epidermal Growth Factor - pharmacology; Fundamental and applied biological sciences. Psychology; Gels; Kinetics; Mice; Mitogens; Molecular Weight; orthovanadate; Phosphatases; Phosphates; Phosphorylation; Protein Kinases - isolation & purification; Protein Kinases - metabolism; protein phosphatase 2A; Ribosomal Protein S6 Kinases; ribosomal proteins; S6 kinase; Transferases; Vanadates; Vanadates - pharmacology; Characterization; Phosphorylation; Regulation(control); Protein kinase; Activation; Identification; Mitogen; Ribosomal protein
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.2.406

Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appears to be distinct from protein kinase C, cAMP-dependent protein kinase, and two protease-activated kinases, PAK II and H4P. The kinase has been purified to apparent homogeneity and migrates as a single band at Mr 70,000 in NaDodSO4/polyacrylamide gels. The kinase exhibits the ability to autophosphorylate, and this activity directly parallels S6 phosphorylation activity on the final step of purification. In vitro, the kinase incorporates up to 5 mol of phosphate into S6, and the tryptic phosphopeptide maps obtained are equivalent to those from S6 phosphorylated in vivo. Most important, treatment of the purified kinase with phosphatase 2A results in complete inactivation of the enzyme, arguing that the activity of the kinase is directly controlled by phosphorylation.