Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells.
In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to -10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M(r)), KP-2 (pI 4.7/20,700 M(r)) and KP-10 (pI 4.7/25,500 M(r)) was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca(2+)-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca(2+)-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.
Nishikawa
K
K
Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
Yamamoto
S
S
Otsuka
C
C
Kato
R
R
D000470:Alkaloids / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D051379:Mice; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D012867:Skin / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D000596:Amino Acids; D010750:Phosphoproteins; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate