Residues 762–801 of PLD1 mediate the interaction with PED/PEA15

Abstract

The interaction of Phospholipase D1 (PLD1) by its C-terminal domain D4 with PED/PEA15 has been indicated as a target for type 2 diabetes. PED/PEA15 is overexpressed in several tissues of individuals affected by type 2 diabetes and its overexpression in intact cells and in transgenic animal models impairs insulin regulation of glucose transport by a mechanism mediated by the interaction with D4 and the consequent increase of protein kinase C-α activity. Expression of D4 or administration of a peptide mimicking the PED/PEA15 region involved in this interaction to cells stably overexpressing PED/PEA15 reduces its interaction with PLD1, thereby lowering PKC-α activation and restoring normal glucose transport mediated by PKC-ζ. By using D4 deletion mutants, we have restricted the PLD1 region involved in PED/PEA15 interaction to an N-terminal fragment named D4α (residues 712–818). This region binds PED/PEA15 with the same efficacy as D4 (K_D ∼ 0.7 μM) and, when transfected in different PED/PEA15-overexpressing cells, it is able to reduce PKC-α activity and to restore the sensitivity of PKC-ζ to insulin stimulation, independently of the PI3K/Akt signalling. We also show that the effective disruption of the PED/PEA15-PLD1 interaction can restore the normal ERK1/2 signalling. Finally, using a set of overlapping peptides that cover the D4α region, we have further restricted the shortest PED/PEA15-binding site to a segment encompassing residues 762–801, suggesting that a quite limited binding interface mostly contributes to the interaction and can thus be a selective target for the design of effective antagonists.