Properties of the 12-O-tetradecanoylphorbol-13-acetate-stimulated S6 kinase from rat astroglial cells.
The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.
Toru-Delbauffe
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D
Unité de Recherche sur la Glande Thyroïde et la Régulation Hormonale, INSERM U.96, Kremlin-Bicêtre, France.
Gavaret
J M
JM
Jacquemin
C
C
Matricon
C
C
Pomerance
M
M
Pierre
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000201:enzymology*; D001921:Brain / Q000201:enzymology*; D002412:Cations; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002412:Cations; D013438:Sulfhydryl Compounds; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide; D004229:Dithiothreitol