Abstract
Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.
Highlights
Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline
Identification of PEA-15 as a plasmids were transfected in a 10:1 (PLD1)-interacting Protein— Mammalian PLD1 contains N-terminal phox and pleckstrin homology domains followed by four conserved regions (I–IV) that are found in all PLD family members
To accomplish the two-hybrid screen, PLD1 was divided into four fragments (F1–F4) encompassing different domains that overlapped in an attempt to preserve the correct three-dimensional structure of each domain (Fig. 1)
Summary
General Reagents—[3H]Dipalmitoyl phosphatidylcholine [cholinemethyl-3H] ([3H]phosphatidylcholine) was obtained from PerkinElmer Life Sciences and palmitic acid [9,10-3H(N)] from American Radiolabeled Chemicals, Inc. Proteins were visualized by Western blotting analysis using monoclonal antibody 3F10 (rat antiHA) and horseradish peroxidase-conjugated secondary antibodies (Roche Molecular Biochemicals). Co-immunoprecipitation—PEA-15 was generated as a GST fusion protein attached to glutathione beads, and FLAG-D4 in phosphatebuffered saline was prepared as described previously [8, 17]. Cells were lysed in 100 l of buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 1ϫ proteinase inhibitor mixture (Roche Molecular Biochemicals), following which the supernatants were added to 100 l of 10% anti-FLAG M2 antibody affinity resin (3.3 mg protein/ml gel, Kodak) that had been washed into the same buffer. Proteins were eluted in 1ϫ sample buffer, and one-quarter of the total elute was subjected to Western analysis to detect the HA tag fused in frame to PEA-15
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