A 20-Amino Acid Module of Protein Kinase Cϵ Involved in Translocation and Selective Targeting at Cell-Cell Contacts

Philippe Clair,Dominique Joubert,Barthélémy Diouf,Nathalie C Guérineau,Frédéric Hollande,Cécile Gauthier-Rouvière,Armelle Choquet,Philippe Jay,Gilles Labesse,Alejandra Collazos,Françoise Macari

doi:10.1074/jbc.m109.004614

Abstract

In the pituitary gland, activated protein kinase C (PKC) isoforms accumulate either selectively at the cell-cell contact (α and ϵ) or at the entire plasma membrane (β1 and δ). The molecular mechanisms underlying these various subcellular locations are not known. Here, we demonstrate the existence within PKCϵ of a cell-cell contact targeting sequence (3CTS) that, upon stimulation, is capable of targeting PKCδ, chimerin-α1, and the PKCϵ C1 domain to the cell-cell contact. We show that this selective targeting of PKCϵ is lost upon overexpression of 3CTS fused to a (R-Ahx-R)<sub>4</sub> (where Ahx is 6-aminohexanoic acid) vectorization peptide, reflecting a dominant-negative effect of the overexpressed 3CTS on targeting selectivity. 3CTS contains a putative amphipathic α-helix, a 14-3-3-binding site, and the Glu-374 amino acid, involved in targeting selectivity. We show that the integrity of the α-helix is important for translocation but that 14-3-3 is not involved in targeting selectivity. However, PKCϵ translocation is increased when PKCϵ/14-3-3 interaction is abolished, suggesting that phorbol 12-myristate 13-acetate activation may initiate two sets of PKCϵ functions, those depending on 14-3-3 and those depending on translocation to cell-cell contacts. Thus, 3CTS is involved in the modulation of translocation via its 14-3-3-binding site, in cytoplasmic desequestration via the α-helix, and in selective PKCϵ targeting at the cell-cell contact via Glu-374.

Highlights

Activation of cytoplasmic kinases often induces their targeting to various subcellular locations where they phosphorylate their substrates and exert their biological functions
In pituitary GH3B6 cells, protein kinase C (PKC) isoforms accumulate at different subcellular locations upon phorbol 12-myristate 13-acetate (PMA) stimulation or thyrotropin-releasing hormone (TRH) receptor activation [10, 11]
To improve our understanding of the molecular mechanisms underlying PKC targeting to the cell-cell contact, we compared the V3 sequences of PKC␣, ⑀, and ␦ [11] with those of PKC␤1 and -␥ (Fig. 1A) and analyzed PKC␤1 and -␥ localization when fused to green fluorescent protein (GFP)

Summary

Introduction

Activation of cytoplasmic kinases often induces their targeting to various subcellular locations where they phosphorylate their substrates and exert their biological functions. To improve our understanding of the molecular mechanisms underlying PKC targeting to the cell-cell contact, we compared the V3 sequences of PKC␣, ⑀, and ␦ [11] with those of PKC␤1 and -␥ (Fig. 1A) and analyzed PKC␤1 and -␥ localization when fused to GFP.

Results

Conclusion