Protein kinase Cdelta as gonadotropin-releasing hormone target isoenzyme in the alphaT3-1 gonadotrope cell line.
We investigated the kinetics of gonadotropin-releasing hormone (GnRH)-induced activation of the protein kinase C (PKC) delta isoform in alphaT3-1 gonadotrope cells. Results were evaluated in subcellular fractions and whole-cell lysates using specific antibodies recognizing either non- or (trans- and auto-)phosphorylated forms of the kinase at Thr505 and Ser643 residues modulating stability and/or activation of the enzyme. Under basal conditions, and in contrast to PKC epsilon, PKC delta was mainly associated with the membrane compartment. GnRH (10(-7)M) elicited further and rapid membrane translocation and time-dependent phosphorylation at both sites of PKC delta. The neuropeptide's effects did not show a refractory period after short but successive GnRH stimulation and were abolished by the GnRH antagonist, antide. Sustained GnRH stimulation (2-6 h) provoked rapid down-regulation of PKC delta. Antide, by inhibiting the initial processes (translocation, phosphorylation), counteracted the degradation of the enzyme. Proteolytic processing of PKC delta was shown to mainly involve proteasome activity. Indeed, specific proteasome inhibitors prevented GnRH-elicited kinase depletion and induced membrane accumulation of the enzyme in a phosphorylated (Thr505, Ser643) form. Thus, GnRH may regulate time-dependent cell responses by modulating the phosphorylation/activation state of its signal transduction effector proteins, and by maintaining their appropriate expression balance via proteolytic processes involving the proteasome system.
Maccario
Hélène
H
CNRS UMR 6544, Université de la Méditerranée, Faculté de Médecine, Marseille, France.
Junoy
Brice
B
Poulin
Benoit
B
Boyer
Bénédicte
B
Enjalbert
Alain
A
Drouva
Sophia V
SV
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D007527:Isoenzymes; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D007987:Gonadotropin-Releasing Hormone; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C497258:PRKCD protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex