The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF.
The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the functionality of the lipase in vivo. We demonstrate that stable short hairpin RNA (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 irrespective 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 with 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. 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.
Di Fulvio
Mauricio
M
Cell Biology and Physiology, Wright State University, School of Medicine, Dayton, OH 45435, USA.
Frondorf
Kathleen
K
Henkels
Karen M
KM
Lehman
Nicholas
N
Gomez-Cambronero
Julian
J
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008049:Lipase / Q000378:metabolism*; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D017931:DNA Primers; D051380:GRB2 Adaptor Protein; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D008049:Lipase; C105606:phospholipase D2; D010739:Phospholipase D