okadaic-acid and iturelix

Protein kinase C (PKC) is a family of isoenzymes playing a key role in the regulation of gonadotrope cell functions. Specific PKC isoforms are activated and downregulated differentially by gonadotropin-releasing hormone (GnRH) and the phorbol ester TPA. In the present study, focusing mainly on PKC epsilon, the mechanisms underlying the proteasome-dependent downregulation of GnRH-activated PKC epsilon and TPA-sensitive PKC alpha and epsilon isoenzymes were investigated in alphaT3-1 gonadotrope cells.. In pull-down assays involving the use of glutathione-agarose affinity beads conjugated with a GST-fusion protein containing ubiquitin-associated domains of Rad23 that bind very likely to K48-linked polyubiquitinated proteins, TPA induced rapid (within 15 min) and sustained (up to 4 h) PKC alpha and PKC epsilon polyubiquitination. However, GnRH selectively elicited receptor-dependent polyubiquitination of PKC epsilon, but not that of PKC alpha. The GnRH-evoked PKC epsilon polyubiquitination was a strong, fast process (taking place as early as 10 min) which decreased progressively with time (but was still detectable after 4 h of treatment). In addition, no apparent association between PKC epsilon and the lysosomal compartment was observed upon performing double-labeling immunofluorescence and confocal microscopy, after either 10 min or 1 hour of stimulation by GnRH or the phorbol ester.. In alphaT3-1 gonadotrope cells, polyubiquitination is therefore the event triggering GnRH-evoked PKC epsilon desensitization as well as TPA-induced PKC alpha and PKC epsilon downregulations; it precedes the respective isoenzyme's degradation by the proteasome complex.

Topics: Animals; Cell Line; Down-Regulation; Gonadotrophs; Gonadotropin-Releasing Hormone; Isoenzymes; Mice; Okadaic Acid; Oligopeptides; Proteasome Endopeptidase Complex; Protein Kinase C-alpha; Protein Kinase C-epsilon; Protein Transport; Tetradecanoylphorbol Acetate; Ubiquitination