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

In the pituitary gland, activated protein kinase C (PKC) isoforms accumulate either selectively at the cell-cell contact (alpha and epsilon) or at the entire plasma membrane (beta 1 and delta). The molecular mechanisms underlying these various subcellular locations are not known. Here, we demonstrate the existence within PKC epsilon of a cell-cell contact targeting sequence (3CTS) that, upon stimulation, is capable of targeting PKC delta, chimerin-alpha 1, and the PKC epsilon C1 domain to the cell-cell contact. We show that this selective targeting of PKC epsilon is lost upon overexpression of 3CTS fused to a (R-Ahx-R)(4) (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 alpha-helix, a 14-3-3-binding site, and the Glu-374 amino acid, involved in targeting selectivity. We show that the integrity of the alpha-helix is important for translocation but that 14-3-3 is not involved in targeting selectivity. However, PKC epsilon translocation is increased when PKC epsilon/14-3-3 interaction is abolished, suggesting that phorbol 12-myristate 13-acetate activation may initiate two sets of PKC epsilon 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 alpha-helix, and in selective PKC epsilon targeting at the cell-cell contact via Glu-374.