Purification of a hepatic S6 kinase from cycloheximide-treated Rats.
Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.
Howard Hughes Medical Institute Laboratories, Harvard Medical School, Boston, Massachusetts.
D J
DJ
Price
R A
RA
Nemenoff
J
J
Avruch
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003513:Cycloheximide / Q000494:pharmacology*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity; D014357:Trypsin
D010446:Peptide Fragments; D003513:Cycloheximide; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D014357:Trypsin; D005033:Ethylmaleimide