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

Abstract

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.

The elucidation of novel SH2 binding sites on PLD2

“…As indicated previously by our laboratory, PLD2 exists as a complex with Grb2 and PTP1b (Horn et al, 2005). Here, we report that PLD2 residues Y 169 and Y 179 directly bind Grb2 and recruit Sos in vivo.…”

“…The data presented here identify for the first time two neighboring tyrosines, Y 169 and Y 179 , in the PLD2 molecule as being essential for the Grb2/PLD2 interaction that our laboratory has described recently (Horn et al, 2005). The amino acids Y 169 and Y 179 in PLD2 are expressed within the context of perfect matches for SH2-mediated Grb2 interaction (pYxNx) (Songyang et al, 1993(Songyang et al, , 1994.…”

“…As for the correlation between PLD2 and tyrosine phosphorylation, it has been previously shown that PLD2 interacts with c-Src and other tyrosine kinases (Slaaby et al, 1998;Ahn et al, 2003;Choi et al, 2004), and the protein tyrosine phosphatase 1b (PTP1b) (Horn et al, 2005). Since the ternary complex PLD2/Grb2/ PTP1b exists in vivo (Horn et al, 2005), it is possible that total tyrosine phosphorylation of PLD2 may also be regulated in a PLD2-Y 179 /Grb2-SH3-dependent manner.…”

Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis

“…Overexpression of XGrb2 SiL did not result in acute upregulation of endogenous Grb2 mRNA due to transfection artifacts, demonstrating that endogenous Grb2 silencing can be rescued at the protein level. Our laboratory has previously shown that Grb2, as part of a Shc-Grb2-SOS complex is essential for the signaling of the phospholipase D2 (PLD2) isoform through the SOS-RAS-RAF-MAPKK-MAPK signaling cascade and eventual effect on DNA synthesis and cell growth [12,13]. Efficient signaling requires association of the lipase with Grb2 through an identified residue, (as the Src homology 'SH' region-2 binds to phosphorylated tyrosine residues).…”

Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner

“…COS7 were routinely transfected following lipofectamine-based procedures as described in detail elsewhere [45]. RAW cells were nucleofected with the Amaxa-based (Gaithersburg, MD) electroporation cuvette with 2×10 6 cells/100 µl in nucleofector solution V. Cells were "zapped" in program D-32 (as per the manufacturer's instructions) after which they were resuspended in 1 ml DMEM + 20% FBS and immediately plated into six-well plates.…”