Ubiquitination as a priming process of PKC alpha and PKC epsilon degradation in the alphaT3-1 gonadotrope cell line.

BACKGROUND/AIM: 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. METHODS/RESULTS: 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. CONCLUSION: 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.

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.

Proteasome implication in phorbol ester- and GnRH-induced selective down-regulation of PKC (alpha, epsilon, zeta) in alpha T(3)-1 and L beta T(2) gonadotrope cell lines.

We investigated mechanisms underlying selective down-modulation of PKC isoforms (alpha, epsilon, zeta): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10(-7) M) or GnRH (10(-7) M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (alpha T(3)-1, L beta T(2)) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in alpha T(3)-1 cells, in a more differentiated gonadotrope cell line L beta T(2) the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (alpha, beta II, delta, epsilon, zeta) were selectively and differentially down-regulated by TPA (alpha, beta II, delta, epsilon) or GnRH (delta, epsilon). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, beta-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC alpha, epsilon, and the GnRH-elicited PKC epsilon down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC alpha, epsilon depletion. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in alpha T(3)-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (alpha, epsilon) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, Gö6976), selectively prevented the TPA-evoked PKC alpha depletion in both mixed pituitary cells and alpha T(3)-1 gonadotropes; in alpha T(3)-1 subcellular fractions, PKC alpha inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses.