Bryostatin 1 protects protein kinase C-delta from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol ester-induced differentiation.
Bryostatin 1 and phorbol-12-myristate-13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in primary mouse keratinocytes bryostatin 1 does not induce differentiation and blocks PMA-induced differentiation. We report here that in primary mouse keratinocytes PMA caused translocation of PKC-epsilon to the Triton X-100-soluble fraction with an approximately 2-order of magnitude higher potency, compared with translocation of PKC-alpha and PKC-delta. The kinetics of translocation were fastest for PKC-epsilon, slower for PKC-alpha, and slowest for PKC-delta. At 5-20 min bryostatin 1 showed potency similar to that of PMA for translocating PKC-alpha, higher potency for translocating PKC-delta, and lower potency for translocating PKC-epsilon. At a later time (6 hr), bryostatin 1 was 1-2 orders magnitude more potent than PMA for causing loss of PKC-alpha, -delta, and -epsilon from the soluble fraction. Bryostatin 1 was 40-fold more potent than PMA for down-regulating PKC-alpha and showed a biphasic dose-response curve for down-regulating PKC-delta. Bryostatin 1 at 0.1-1 nM down-regulated PKC-delta to a similar extent as did PMA. Bryostatin 1 at 100 nM to 1 microM, on the other hand, failed to induce down-regulation, and these high (100 nM to 1 microM) doses of bryostatin 1 showed noncompetitive inhibition of PKC-delta down-regulation by 1 microM PMA after coapplication. This protected portion of PKC-delta retained kinase activity. The dose-response curve for bryostatin 1 protection of PKC-delta from down-regulation by PMA correlated with bryostatin 1 inhibition of the effects of PMA on cornified envelope formation (a marker of differentiation) and epidermal growth factor binding. Although PKC-epsilon was readily translocated by both PMA and bryostatin 1, the PKC-epsilon originally associated with the particulate fraction showed no down-regulation by either of these agents. We hypothesize that differential regulation of PKC isozymes by PMA and bryostatin 1 may contribute to the different patterns of biological responses that they induce.
Szallasi
Z
Z
Laboratory of Cellular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Denning
M F
MF
Smith
C B
CB
Dlugosz
A A
AA
Yuspa
S H
SH
Pettit
G R
GR
Blumberg
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D054713:Bryostatins; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D007527:Isoenzymes / Q000187:drug effects*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000187:drug effects*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors*
D000970:Antineoplastic Agents; D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D011500:Protein Synthesis Inhibitors; C046785:bryostatin 1; C497259:Prkcd protein, mouse; C497256:Prkce protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate