The Grb2/PLD2 Interaction Is Essential for Lipase Activity, Intracellular Localization and Signaling in Response to EGF

Abstract

The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the lipase's functionality in vivo. In this study, we demonstrate that stable shRNA-based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2 SiL ) into the Grb2-knockdown cells (COS7 shGrb2 ), resulted in the nearly full rescue of PLD2 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7 shGrb2 cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespectively of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments of Grb2 and PLD2-Y169/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGF. In summary, these results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo.

Phosphatidic Acid Is a Leukocyte Chemoattractant That Acts through S6 Kinase Signaling

“…Fresh DMSO (1.25% (v/v) was added to the media after nucleofection, and cells were cultured for an additional 48-h period. In selected experiments, cells were overexpressed with a Grb2 plasmid (pcDNA-XGrb2) (17). The expression construct containing the full cDNA of human Grb2 variant 1 (pCMV6-Grb2, OriGene Technologies) was the template for PCR subcloning.…”

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

“…Transient expression of mycPLD2 Y179F increases p42/44 ERK phosphorylation [25,26] and AKT crosstalk with Raf modulating its function as a Mek activator [29,54,55]. MycPLD2 Y179F-mediated activation of p42/44 ERK was dependent on PI3K activity suggesting that PI3K/AKT is located upstream of p42/44 ERK .…”

“…In order to investigate if PLD2 would modulate cell survival and proliferation through the AKT/PI3K pathway, we transiently transfected COS7 cells with PLD2 Y179F, a mutant known to increase DNA synthesis in this cell line [25,26]. The expression levels and localization of PLD2 WT did not differ from PLD2 Y179F, as shown in Figure 1A.…”

“…Expression of pcDNA-mycPLD2 (WT or Y179F), were analyzed by immunofluorescence following procedures as described in detail elsewhere [26]. Cells were treated with 2 µg/ml anti-myc antibodies and with 0.5 µg/ml FITC-conjugated secondary antibody and, lastly, with DAPI for nuclear staining.…”

“…PLD may participate in the activation of p42/44 ERK via Raf-1 translocation to the plasma membrane [20][21][22][23], as well as in the survival signaling mediated by PI3K/AKT activation [24]. Although the precise mechanisms involving the isoform PLD2 in cell proliferation and survival are still unknown, recent evidence suggests that residues Y 169 and Y 179 serve to recruit the Grb2/Sos complex and that PLD2-Y 169 is involved in Ras/ MAPK activation [25,26].…”