A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C.
The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.
Deutsches Krebsforschungszentrum, Institut für Biochemie, Heidelberg, Federal Republic of Germany.
M
M
Gschwendt
W
W
Kittstein
F
F
Horn
H
H
Leibersperger
F
F
Marks
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004817:Epidermis / Q000201:enzymology*; D005260:Female; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D010703:Phorbol Esters / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010703:Phorbol Esters; D010743:Phospholipids; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium