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      Protein Kinase Cδ as Gonadotropin-Releasing Hormone Target Isoenzyme in the αT3-1 Gonadotrope Cell Line

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          Abstract

          We investigated the kinetics of gonadotropin-releasing hormone (GnRH)-induced activation of the protein kinase C (PKC) δ isoform in αT3-1 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 Ε, PKC δ was mainly associated with the membrane compartment. GnRH (10<sup>–7</sup> M) elicited further and rapid membrane translocation and time-dependent phosphorylation at both sites of PKC δ. 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 δ. Antide, by inhibiting the initial processes (translocation, phosphorylation), counteracted the degradation of the enzyme. Proteolytic processing of PKC δ 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.

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          Most cited references 36

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          Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions.

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            Ubiquitin in chains

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              Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms.

              The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1). In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1-/- cells), the intracellular levels of endogenously expressed PKCalpha, PKCbetaI, PKCgamma, PKCdelta, PKCepsilon, and PKC-related kinase-1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCzeta and PRK2 protein are only moderately reduced in the PDK1-/- ES cells. We demonstrate that in contrast to PKCzeta expressed PDK1+/+ ES cells, PKCzeta in ES cells lacking PDK1 is not phosphorylated at its T-loop residue. This provides the first genetic evidence that PKCzeta is a physiological substrate for PDK1. In contrast, PRK2 is still partially phosphorylated at its T-loop in PDK1-/- cells, indicating the existence of a PDK1-independent mechanism for the phosphorylation of PRK2 at this residue.
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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                2004
                May 2004
                10 June 2004
                : 79
                : 4
                : 204-220
                Affiliations
                CNRS UMR 6544, Université de la Méditerranée, Faculté de Médecine, Marseille, France
                Article
                78102 Neuroendocrinology 2004;79:204–220
                10.1159/000078102
                15153754
                © 2004 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 9, References: 64, Pages: 17
                Categories
                Regulation of Pituitary Cells

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