Abstract
Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.
Highlights
Population of type 2 diabetic individuals and their first degree relatives (7, 8)
Transgenic mice for PED/PEA-15 display impaired glucose tolerance and develop diabetes, if fed a high fat diet (9). All these observations suggest that PED/PEA-15 overexpression could be involved in the complex series of events leading to type 2 diabetes, one of the most common disorder in the world associated with impaired insulin action and secretion and for which no single defect has been so far unequivocally determined (12–14)
Two consensus serine phosphorylation sites have been identified at the C terminus of the protein (Ser[104] and Ser116), and phosphorylation by protein kinase C (PKC),[3] calmodulin kinase II, and AKT/protein kinase B has been shown to occur in different cells types and to contribute to the regulation of PED/PEA-15 protein stability (16 –18)
Summary
PcDNA3 Vector Preparation—Cloning of PED/PEA-15 cDNA in pcDNA3 was previously described (22). PED/PEA-15 was further purified on a 1-ml Mono Q HR 5/5 column (GE Healthcare) applying a gradient from 0% to 100% NaCl in 50 mM Tris-HCl, 500 mM NaCl, pH 7.5, buffer. PED/PEA-15 Tryptic Digestion—PED/PEA-15 proteolysis was performed by digestion with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma-Aldrich) at an enzyme:substrate ratio of 1:100 (w/w) in 50 mM Tris-HCl, pH 7.5, for 16 h. Mass calibration was PEA-15 peptides, 1 M Trx-His6-D4 was preincubated with 30 performed automatically by means of selected multiple charged M of each peptide at 25 °C for 30 min before injection. After trifluoroacetic acid-H2O-triisopropylsilane mixture (500 washing with PBS-NaCl buffer (300 mM NaCl, 2.7 mM KCl, 10 l/100 mg of resin) and purified by RP-HPLC. Following 1-h incubation in the dark at 37 °C, solutions were removed, and wells were again washed with PBS-NaCl buffer.
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