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-alpha 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-alpha activation and restoring normal glucose transport mediated by PKC-zeta. By using D4 deletion mutants, we have restricted the PLD1 region involved in PED/PEA15 interaction to an N-terminal fragment named D4alpha (residues 712-818). This region binds PED/PEA15 with the same efficacy as D4 (K(D) approximately 0.7 microM) and, when transfected in different PED/PEA15-overexpressing cells, it is able to reduce PKC-alpha activity and to restore the sensitivity of PKC-zeta 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 D4alpha 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.

Discovery of Small Peptide Antagonists of PED/PEA15–D4α Interaction from Simplified Combinatorial Libraries

“…This K D is in very good agreement with that previously reported (22) and supports the view that the binding of PLD1 to PED/PEA15 is essentially mediated by the region D4 α‐spanning residues 712–818.…”

PEA-15 potentiates H-Ras-mediated epithelial cell transformation through phospholipase D

“…These findings in addition to our finding that PLD1 co-localizes with PEA-15 in iBMK cells raised the question if binding of the two proteins is necessary for the observed enhancement of ERK activity in H-Ras expressing iBMK cells. The PEA-15 binding region of PLD1 has already been mapped to a C-terminal domain D4 (AA 712-1074) and the residues 762-801 within this domain have been identified as the shortest PEA-15 binding segment (Figure 7A) (Doti et al 2010, Zhang et al 2000). Here we used a PLD1 construct only consisting of the D4 domain (Figure 7B) and lacking catalytic activity (PLD1-D4).…”

Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

“…Together with the positive effect of D4 expression on glucose-induced insulin secretion, this may contribute to the amelioration of whole body glucose homeostasis. Recently, Doti N. et al have shown that the use of a restricted D4 domain, named D4alpha, or the use of short PLD1 peptides that could mimic PED/PEA-15 binding interface can be used as antagonists to prevent the association of PLD1 from PED/PEA-15 [24]. This study provided thus new short, selective and efficient PED/PEA-15-PLD1 antagonists alternative to the D4 that could be transduced to the cells and tissues by adenoviral gene delivery approach.…”