title: Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form.
abstract: Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Gould
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Corina E
authors.initials: CE
authors.lastname: Antal
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Gloria
authors.initials: G
authors.lastname: Reyes
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Maya T
authors.initials: MT
authors.lastname: Kunkel
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Adams
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Ahdad
authors.initials: A
authors.lastname: Ziyar
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721; Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093-0721.
authors.forename: Tania
authors.initials: T
authors.lastname: Riveros
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0721. Electronic address: anewton@ucsd.edu.
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000502:physiology*; D002522:Chlorocebus aethiops; D004795:Enzyme Stability / Q000187:drug effects / Q000235:genetics; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase
chemical_list: D047428:Protein Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C