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Characterization of a phorbol ester-stimulated S6 kinase from MDCK renal epithelial cells

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6015402
;
Increased phosphorylation of S6, a 40S ribosomal subunit protein, is observed in mammalian cells in response to growth factors and phorbol esters. The goal of this study was to identify the S6 kinase that is stimulated by phorbol ester treatment of MDCK cells. MDCK clone D1 cells express high levels of protein kinase C(PKC). PKC and S6 kinase activities were measured following DEAE-Sephacel fractionation of cytosol; this procedure separated the two kinase activities. When confluent MDCK-D1 cells were exposed to 100 nM phorbol 12-myristate 13-acetate (PMA), 95% of the total cellular PKC activity became associated with the particulate fraction within 1 hour. Cytosolic S6 kinase activity was maximal by 1 hour and then declined thereafter, preceding any detectable loss of total cellular PKC. The PMA-responsive S6 kinase was partially purified from MDCK-D1 cytosol by consecutive steps of DEAE-Sephacel, ammonium sulfate precipitation, Ultrogel AcA 34, heparin-agarose, and Ultrogel AcA 34. The partially-purified enzyme had an apparent molecular size of approximately 80 kDa. In addition to S6, the enzyme phosphorylated synthetic peptides based on the carboxyl terminal sequence of S6. S6 kinase activity utilized ATP but not GTP, and was inhibited by heparin, NaCl, and ..beta..-glycerophosphate. In conclusion, a phorbol ester-stimulated S6 kinase has been partially purified from an epithelial cell line. This kinase is distinct from PKC.
Research Organization:
Howard Hughes Medical Institute, Seattle, WA
OSTI ID:
6015402
Report Number(s):
CONF-870644-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
Country of Publication:
United States
Language:
English

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Related Subjects

560300* -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL TISSUES
BIOLOGICAL EFFECTS
BODY
CARCINOGENS
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
ENZYME ACTIVITY
ENZYMES
EPITHELIUM
ESTERS
FRACTIONATION
KIDNEYS
MOLECULAR WEIGHT
ORGANIC COMPOUNDS
ORGANS
PHORBOL ESTERS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
SEPARATION PROCESSES
TISSUES
TRANSFERASES