GTP-bound Ras was affinity-purified from 500 g of COS7 lysates transiently expressing mycPLD2 WT or Y169/Y179 mutants by using the Ras-binding domain (RBD) of Raf1 coupled to agarose beads (RBD-Raf1-Agarose, Upstate, NY). to engage the EGFR with cell proliferation direct recruitment and activation of the MAPK signaling pathway9. Stimulation of COS7 cells with EGF causes the cytosolic Grb2 to partially relocate to LY 344864 S-enantiomer the plasma membrane (the place where the interaction of EGFR to Grb2 occurs) which is followed by localization in vesicle-like structures at the perinuclear region of the Golgi apparatus15; 16. An intracellular relocalization of PLD2 followed EGF treatment has been explored before in mouse17; 18; 19, but a connection between PLD2 and Grb2 has not been addressed experimentally as of yet. Our laboratory has recently demonstrated that PLD2 interacts with the Grb2/Sos complex5; 20, and here we sought to ascertain how critical the presence of Grb2 was for the functionality (activity and intracellular localization) of the lipase transient transfection of antibody. As shown in Figure 1D, XGrb2SiL transiently expressed in COS7shGrb2 cells (clone #4) localizes diffusely in the cytoplasm. This is in agreement to other authors findings for different Grb2 constructs in COSWT cells16. Taken together, these results demonstrate that endogenous Grb2 expression can be specifically knocked-down and rescued in COS7shGrb2 cells. Open in a separate window Figure 1 Expression of Grb2 in COS7shGrb2 cells(A) Western blot estimation of endogenous Grb2 expression (~25 kDa) in COS7 cells wild-type (COS7WT) and four different clones of stable transfected cells (COS7shGrb2 or COS7shControl). As a loading Control, a b-actin blot of the transferred membrane is also shown. (B) Densitometric analysis of endogenous Grb2 expression in COS7WT, COS7shGrb2 and COS7shControl cells expressed as mean SEM. At least 5 different immunoblots were analyzed (*p 0.001). (C) Silenced expression of endogenous Grb2 in COS7shGrb2 cells is rescued by transient transfection of a silent version of Grb2 (XGrb2SiL). COS7shGrb2 cells were transfected with 2 mg of pcDNA-XGrb2SiL WT and the expression of endogenous Grb2 (~25 kDa) and transfected XGrb2SiL (~30 kDa) were analyzed by Western blot using an anti-Grb2 antibody. Shown is a representative experiment. (D) Direct immunofluorescent detection of XGrb2SiL WT in COS7shGrb2 cells. COS7shGrb2 cells transiently expressing pcDNA-XGrb2SiL for 48 hours were serum-starved overnight and analyzed for XGrb2SiL localization by direct immunofluorescence using a FITC-conjugated antibody against the antibody. PLD2 WT, K758R and endogenous Grb2 expression levels determined by myc or Grb2-immunoblot of whole COS7 cell lysates. (B, PLD2 interaction with the Grb2/Sos complex analyzed by co-immunoprecipitation. As shown in Figure 4A, PLD2 WT interacts with Grb2/Sos, as demonstrated previously5; 20. However, PLD2 Y169/179F mutant was unable to co-immunoprecipitate the Grb2/Sos complex. Moreover, PLD2 Y169/179F was unable to activate Ras or p42/44ERK, suggesting that Grb2 binding to PLD2 plays an essential role in MAPK activation (Figure 4B). Simultaneously, COS7WT lysates transiently expressing mycPLD2 WT, Y169/179F or K758R were analyzed for total enzymatic activity. As shown in Figure 4C, PLD2 Y169/179F is catalytically inactive, suggesting that PLD2 association to Grb2 is involved in high basal PLD2 activity. Moreover, Grb2 association to PLD2 appear to be independent of EGFR stimulation of COS7shGrb2 cells (Figure 5). Open in a separate window Figure 4 Human PLD2 Y169/179F is a catalytically inert enzyme which cannot interact with the Grb2/Sos complex or activate Ras (A) Co-immunoprecipitation of mycPLD2 and/or Grb2 TRAILR4 from COS7WT cells transiently expressing pcDNA-mycPLD2 WT or Y169/179F. COS7WT cells were transiently transfected with the indicated plasmids and after 48 hours, association of mycPLD2 with the Grb2/Sos complex analyzed by co-immunoprecipitation using myc or Grb2 antibodies. The immunological presence of mycPLD2, Grb2 and Sos in myc-immunoprecipitates are shown together with the expression of each protein in whole COS7WT lysates. (B) Activation LY 344864 S-enantiomer of the Ras/MAPK signaling pathway was analyzed by direct determination of active Ras (immunoipurified GTP-bound Ras) and by the degree of p42/44ERK phosphorylation in residues T/Y using immunoblot. Ras/MAPK activation was analyzed in whole lysates of COS7WT cells transiently expressing pcDNA-mycPLD2 WT, Y169/179F or Y179F (positive Control) for 48 hours. (C, (Figure 4), could not cause an LY 344864 S-enantiomer intracelluar redistribution of Grb2 in response to EGF as mycPLD2 WT did in COS7shGrb2 cells co-expressing XGrb2SiL (Figure 7). Hence, LY 344864 S-enantiomer these results indicate that mycPLD2 localization in the preinuclear Golgi region in EGF-stimulated cells depends on Grb2, probably SH2-mediated interaction.