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

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Phagocytosis is a primary innate response of both macrophages and neutrophils involving the formation of filamentous actin (F-actin)-rich protrusions that are extended around opsonized pathogens to form a phagocytic cup, resulting in their subsequent internalization. The molecular mechanism for this is still not completely understood. We now show for the first time that phospholipase D2 (PLD2) binds to growth factor receptor-bound protein 2 (Grb2) and to the Wiskott-Aldrich syndrome protein (WASp) to form a heterotrimer complex, PLD2-Grb2-WASp, and present the mechanism of interaction. Grb2 binds to the Y169/Y179 residues of PLD2 using its only SH2 domain, and it interacts with the poly-proline region of WASp using its two SH3 domains. The PLD2-Grb2-WASp heterotrimer can be visualized in early phagocytic cups of macrophages ingesting opsonized red blood cells, where it associates with polymerized actin. Cup colocalization and phagocytosis are disrupted with mutants that alter binding at either of the two proteins or by silencing Grb2 with RNA interference (RNAi). WASp association to PLD2-K758R, a lipase-inactive mutant, still occurs, albeit at lower levels, indicating that PLD2 plays a second role in phagocytosis, which is the production of phosphatidic acid (PA) and activation of phosphatidylinositol 5-kinase (PI5K) with subsequent synthesis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The latter can be blocked with RNAi, which negates phagocytosis. Lastly, a constitutively "open" active form of WASp (WASp-L270P) brings phagocytosis to its maximum level, which can be mimicked with WASp-WT plus PLD2 or plus PA. Since neither a protein-protein disruption nor lack of PLD activity completely negates cup formation or phagocytosis, we posit a two-step mechanism: PLD2 anchors WASp at the phagocytic cup through Grb2 following protein-protein interactions and also activates it, making key lipids available locally. The heterotrimer PLD2-Grb2-WASp then enables actin nucleation at the phagocytic cup and phagocytosis, which are at the center of the innate immune system function.

Section: Discussionmentioning

confidence: 94%

A Novel Phospholipase D2-Grb2-WASp Heterotrimer Regulates Leukocyte Phagocytosis in a Two-Step Mechanism

Kantonen

Hatton

Mahankali

et al. 2011

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“…The expression plasmid encoding the catalytically inactive form of SHP-2, SHP-2CA, was previously described (26). The Grb2 short hairpin RNA (shRNA) construct (pU6 ϩ27 -ShGrb2) directed against Grb2 nucleotides 310 to 330 (GAT GTG CAG CAC TTC AAG GTT) used for knockdown studies is as described previously (8). The strategy to generate the Grb2YF form unrecognizable by the Grb2 shRNA, the Sil-Grb2YF construct, is as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…The Grb2 short hairpin RNA (shRNA) construct (pU6 ϩ27 -ShGrb2) directed against Grb2 nucleotides 310 to 330 (GAT GTG CAG CAC TTC AAG GTT) used for knockdown studies is as described previously (8). The strategy to generate the Grb2YF form unrecognizable by the Grb2 shRNA, the Sil-Grb2YF construct, is as described previously (8). This was achieved by incorporating seven silent mutations in the myc-Grb2YF plasmid within the region targeted by Grb2 shRNA (GAC GTC CAA CAT TTT AAA GTA; the silent mutations are underlined) (GenScript Corporation, Piscataway, NJ).…”

Section: Methodsmentioning

confidence: 99%

Tyrosine Phosphorylation of Grb2: Role in Prolactin/Epidermal Growth Factor Cross Talk in Mammary Epithelial Cell Growth and Differentiation

Haines

Minoo

Feng

et al. 2009

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Characterizing mechanisms regulating mammary cell growth and differentiation is vital, as they may contribute to breast carcinogenesis. Here, we examine a cross talk mechanism(s) downstream of prolactin (PRL), a primary differentiation hormone, and epidermal growth factor (EGF), an important proliferative factor, in mammary epithelial cell growth and differentiation. Our data indicate that EGF exerts inhibitory effects on PRL-induced cellular differentiation by interfering with Stat5a-mediated gene expression independent of the PRL-proximal signaling cascade. Additionally, our data show that PRL is a potent inhibitor of EGF-induced cell proliferation. We identify tyrosine phosphorylation of the growth factor receptor-bound protein 2 (Grb2) as a critical mechanism by which PRL antagonizes EGF-induced cell proliferation by attenuating the activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. Together, our results define a novel negative cross-regulation between PRL and EGF involving the Jak2/Stat5a and Ras/MAPK pathways through tyrosine phosphorylation of Grb2.

“…We have recently found that Fes binds PA and participates in a PLD-induced pathway of myeloid differentiation (15). PLD2 is associated with EGFR signaling by binding to Grb2 at two specific residues (Y169 and Y179) that leads to activation of the growth factor pathway (16)(17)(18). PA interacts with Sos during EGF-induced membrane recruitment and Ras activation (6).…”

mentioning

confidence: 99%

Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor

Hatton

Lintz

Mahankali

et al. 2015

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Overexpression of epidermal growth factor receptor (EGFR) is one of the frequent mechanisms implicated in cancer progression, and so is the overexpression of the enzyme phospholipase D (PLD) and its reaction product, phosphatidic acid (PA). However, an understanding of how these signaling molecules interact at the level of gene expression is lacking. Catalytically active PLD enhanced expression of EGFR in human breast cancer cells. Overexpression of the PLD2 isoform increased EGFR mRNA and protein expression. It also negated an EGFR downregulation mediated by small interfering RNA targeting EGFR (siEGFR). Several mechanisms contributed to the alteration in EGFR expression. First was the stabilization of EGFR transcripts as PLD2 delayed mRNA decay, which prolonged their half-lives. Second, RNase enzymatic activity was inhibited by PA. Third, protein stabilization also occurred, as indicated by PLD resistance to cycloheximide-induced EGFR protein degradation. Fourth, PA inhibited lysosomal and proteasomal degradation of internalized EGFR. PLD2 and EGFR colocalized at the cell membrane, and JAK3 phosphorylation at Tyr980/Tyr981 followed receptor endocytosis. Further, the presence of PLD2 increased stabilization of intracellular EGFR in large recycling vesicles at ϳ15 min of EGF stimulation. Thus, PLD2-mediated production of PA contributed to the control of EGFR exposure to ligand through a multipronged transcriptional and posttranscriptional program during the out-of-control accumulation of EGFR signaling in cancer cells. Epidermal growth factor receptor (EGFR) is overexpressed in many epithelial tumors, including bladder, kidney, pancreatic, and squamous cell carcinomas (1). In breast cancer, EGFR overexpression is associated with advanced-stage disease and shortened relapse-free survival, which occurs concomitantly with low estrogen receptor expression (2). The cell signaling events after EGFR ligand stimulation and cancer have been greatly studied and require association of the receptor with a number of cytoplasmic tyrosine kinases, as well as activation of the Janus kinase (JAK)/ STAT pathway (3). EGFR directly interacts with phospholipase D2 (PLD2) (4-6). Stimulation of EGFR increases cellular PLD activity and the production of phosphatidic acid (PA) in cancer cell lines (7,8). PA is also the precursor to lysophosphatidic acid (LPA) that is relevant in ovarian cancer (9). PLD2 activity is regulated by the phosphorylation of the kinases EGFR and Janus kinase 3 (JAK3) on the Y296 and Y415 tyrosine residues, respectively (10, 11). JAK3 is a 130-kDa intracellular, nonreceptor tyrosine kinase (12, 13). It can also function as the docking site for other proteins if they have the proper Src homology 2 (SH2) domains.Tyrosine kinases associated with EGFR, such as Fer/Fes, promote cell motility in a PLD/PA-dependent pathway (14). We have recently found that Fes binds PA and participates in a PLD-induced pathway of myeloid differentiation (15). PLD2 is associated with EGFR signaling by binding to Grb2 at two specific res...