The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2.
The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.
Horn
Jeff
J
Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA.
Lopez
Isabel
I
Miller
Mill W
MW
Gomez-Cambronero
Julian
J
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D051380:GRB2 Adaptor Protein; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D046912:Multiprotein Complexes / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D046912:Multiprotein Complexes; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C105606:phospholipase D2; D010739:Phospholipase D