Insulin-sensitive, serum-sensitive protein kinase activity that phosphorylates ribosomal protein S6 in cultured fibroblast-melanoma hybrid cells.
A protein kinase activity (S6PK) that phosphorylates ribosomal protein S6 has been detected in cytosolic extracts prepared from an insulin-sensitive mouse fibroblast-melanoma hybrid cell line. The activity of this enzyme is greatly increased in cells that have been stimulated with insulin or serum for 30 min before preparation of the extract. In the parental melanoma cells, which are insensitive to the growth-stimulatory action of insulin, the activity of the enzyme is lower than in the hybrid cells and is not increased in response to insulin. The insulin-sensitive, serum-sensitive S6PK from the hybrid cells is eluted as a single peak from diethylaminoethyl (DEAE)-cellulose between 0.15 and 0.2 M KCl. The apparent mol wt of the enzyme, as determined by gel permeation chromatography, is approximately 105,000. A second S6 kinase activity from the hybrid cells is trypsin dependent and elutes from DEAE-cellulose at a lower salt concentration than S6PK. In contrast to S6PK, the trypsin-dependent S6 kinase activity does not vary in a consistent manner in response to insulin or serum. Fractions obtained from DEAE-cellulose chromatography of extracts of the hybrid cells have also been assayed for ability to phosphorylate the synthetic octapeptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6-1), the structure of which is based on a phosphorylated region of the S6 protein. Two trypsin-dependent peaks of protein kinase activity have been found to phosphorylate this peptide, one eluting at 0.05 M KCl and the other at 0.10-0.15 M KCl. The first peak elutes at the same salt concentration as the trypsin-dependent protein kinase(s) that phosphorylate ribosomal protein S6, while the second elutes slightly, but reproducibly ahead of S6PK. Several properties of the second peak of S6-1 phosphorylating activity suggest that it is not S6PK.
D000818:Animals; D001769:Blood; D002455:Cell Division; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose; D005347:Fibroblasts / Q000201:enzymology*; D006822:Hybrid Cells / Q000201:enzymology*; D007328:Insulin / Q000494:pharmacology*; D008545:Melanoma / Q000201:enzymology*; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D007328:Insulin; D009842:Oligopeptides; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D011494:Protein Kinases; D011493:Protein Kinase C; D014357:Trypsin