The catalytic domain of protein kinase Cdelta confers protection from down-regulation induced by bryostatin 1.
Bryostatin 1 (Bryo) has been shown to induce biphasic dose-response curves for down-regulating protein kinase Cdelta (PKCdelta) as well as for protecting PKCdelta from down-regulation induced by phorbol 12-myristate 13-acetate (PMA). To identify regions within PKCdelta that confer these responses to Bryo, we utilized reciprocal PKCalpha and PKCdelta chimeras (PKCalpha/delta and PKCdelta/alpha) constructed by exchanging the regulatory and catalytic domains of these PKCs. These chimeras and wild-type PKCalpha/alpha and PKCdelta/delta constructed in the same way were stably expressed in NIH 3T3 fibroblasts. Twenty-four h of treatment with Bryo induced a biphasic dose-response curve for down-regulating both wild-type PKCdelta/delta and the PKCalpha/delta chimera. In contrast, Bryo led to a nearly complete down-regulation of both PKCalpha/alpha and PKCdelta/alpha and also produced a faster mobility form of these species on SDS-polyacrylamide gel electrophoresis. The nature of both the regulatory and, to a lesser extent, the catalytic domains affected the potency of Bryo to down-regulate the chimeric PKC proteins as well as to protect PKCalpha/delta and PKCdelta/delta from down-regulation. Bryo at high concentrations also inhibited the down-regulation of PKCdelta/delta and PKCalpha/delta induced by 1 microM PMA when co-applied. The portion of PKC protected by Bryo from down-regulation by either Bryo or PMA was localized in the particulate fraction of the cells. We conclude that the catalytic domain of PKCdelta confers protection from down-regulation induced by Bryo or Bryo plus PMA, suggesting that this domain contains the isotype-specific determinants involved in the unique effect of Bryo on PKCdelta.
Lorenzo
P S
PS
Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Bögi
K
K
Acs
P
P
Pettit
G R
GR
Blumberg
P M
PM
D016475:3T3 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D054713:Bryostatins; D002384:Catalysis; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D051379:Mice; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000970:Antineoplastic Agents; D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C046785:bryostatin 1; C497259:Prkcd protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate