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

Abstract

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...

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

“…We have also seen in Figures 3, 4 and 5 a greater accumulation of PA in vesicles around the nucleus and that this surge in PA led to an increase in EGFR expression, leading us to investigate the nuclear mechanism by which PA increases EGFR expression. PA localizes to the nucleus and interacts with nuclear receptors [39–41]. We have shown previously that PA alters the secondary structure of PPARα [41].…”

“…Both EGFR and PA are transferred to the nucleus to influence gene expression, and some to the Golgi for recycling/export back out of the cell. PA in the nucleus activates gene expression of several genes including EGFR, as PA is a mitogen [39]. Newly synthesized EGFR is then shuttled through the Golgi to the plasma membrane through vesicles.…”

Phospholipase D2 promotes disease progression of renal cell carcinoma through the induction of angiogenin

“…The phospholipid 1,2,‐dioleyl phosphatidic acid (DOPA) was purchased from Avanti Polar Lipids (840875P, Alabaster, AL, USA). Lipid preparation was carried out as described previously . Briefly, DOPA was prepared from powder in PBS with 0.5% BSA for a final concentration of 1 mmol/L.…”

“…Lipids were added to the cells for a final concentration of 300 nmol/L for 4 hours. Previous reports have shown that this form of PA is cell soluble . Post‐treatment cells were harvested and subjected to quantitative RT‐PCR (qRT‐PCR).…”

Loss of Diacylglycerol Kinase-Ζ Inhibits Cell Proliferation and Survival in Human Gliomas

“…It has been reported that PA, as the product of phospholipase D (PLD), is critical for cancer cell survival and progression, the activity of which is enhanced in multiple cancer types [7,8]. PA also could be generated by other several metabolic pathways including diacylglycerol kinases (DGKs) and lysophosphatidic acid acyltransferase (LPAAT) [9].…”