Residues 762-801 of PLD1 mediate the interaction with PED/PEA15.
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.
Doti
Nunzianna
N
Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134 Napoli, Italy.
Cassese
Angela
A
Marasco
Daniela
D
Paturzo
Floriana
F
Sabatella
Marco
M
Viparelli
Francesca
F
Dathan
Nina
N
Monti
Simona M
SM
Miele
Claudia
C
Formisano
Pietro
P
Beguinot
Francesco
F
Ruvo
Menotti
M
D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction
D051017:Apoptosis Regulatory Proteins; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C092039:PEA15 protein, human; D010750:Phosphoproteins; D010739:Phospholipase D; C106801:phospholipase D1