The roles of phospholipase D in EGFR signaling

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/ S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nM rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/ S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration. Phospholipase D (PLD)3 catalyzes the hydrolysis of the terminal diester bond of glycerophospholipids resulting in the formation of phosphatidic acid (PA) plus a related base in cell membranes. Two different genes encoding for mammalian phospholipase D exist: PLD1 and PLD2 (1-2). PLD-generated PA is a second messenger involved in mitogenesis, cell growth, and migration (3-6). Most significant effects of elevated PLD activity are mediated by targets of PA (6). Although several of these have been identified (7), we will concentrate on two of them, the mammalian target of rapamycin (mTOR) (8) and S6 kinase (S6K) (9). PA binds mTOR and activates it (8, 10). PA also binds and activates S6K independently of mTOR (9). Regardless, mTOR and/or S6K phosphorylates downstream targets resulting in the modulation of diverse cellular functions such as survival, cell migration and growth, and proliferation (6, 8, 10 -12).IL-8 is a potent chemoattractant that elicits cell migration in neutrophilic HL-60 cells (13), T lymphocytes, monocytes, and natural killer cells (14 -18). The PLD2 isozyme appears to be a functional target of IL-8-mediated activation of both CXCR1 and CXCR2, the two known receptors for 19). PLDderived PA mediates cell migration as well as mTOR/S6K activation (8 -10, 14). Indeed, the relevance of mTOR in cell migration and inflammation has been recently reported for T lymphocytes (20), macrophages (21), and neutrophils (22). Furthermore, leukocyte chemotaxis is inhibited by the macrolide immunosuppressant rapamycin, a specific inhibitor of the mTOR/S6K pathway (23).The impact of the mTOR/S6K signaling cascade and the role of this pathway on PLD function have not been investigate...

mentioning

confidence: 99%

Mammalian Target of Rapamycin (mTOR) and S6 Kinase Down-regulate Phospholipase D2 Basal Expression and Function

Tabatabaian¹,

Dougherty²,

Fulvio

et al. 2010

“…4, B and C). There is evidence suggestive of aberrant PLD activity in cells transformed by v-Src (34), H-Ras (35), and v-Raf (36), as well as evidence for the overexpression of PLD in cancer tissues (37). In the presence of 0.5% serum, the aggressive breast cancer cell MDA-MB-231 shows higher PLD activity than that of another relatively benign breast cancer cell line, MCF-7 (38).…”

Section: Discussionmentioning

confidence: 98%

Heterogeneity of Phosphatidic Acid Levels and Distribution at the Plasma Membrane in Living Cells as Visualized by a Förster Resonance Energy Transfer (FRET) Biosensor

Nishioka

Frohman

Matsuda

et al. 2010

Phosphatidic acid (PA) is one of the major phospholipids in the plasma membrane. Although it has been reported that PA plays key roles in cell survival and morphology, it remains unknown when and where PA is produced in the living cell. Based on the principle of Förster resonance energy transfer (FRET), we generated PA biosensor, and named Pii (phosphatidic acid indicator). In these biosensors, the lipid-binding domain of DOCK2 is sandwiched with the cyan fluorescent protein and yellow fluorescent protein and is tagged with the plasma membrane-targeting sequence of K-Ras. The addition of synthetic PA, or the activation of phospholipase D or diacylglycerol kinase at the plasma membrane, changed the level of FRET in Pii-expressing cells, demonstrating the response of Pii to PA. The biosensor also detected divergent PA content among various cell lines as well as within one cell line. Interestingly, the growth factor-induced increment in PA content correlated negatively with the basal PA content before stimulation, suggesting the presence of an upper threshold in the PA concentration at the plasma membrane. The biosensor also revealed uneven PA distribution within the cell, i.e. the basal level and growth factorinduced accumulation of PA was higher at the cell-free edges than at the cell-cell contact region. An insufficient increase in PA may account for ineffective Ras activation at areas of cell-cell contact. In conclusion, the PA biosensor Pii is a versatile tool for examining heterogeneity in the content and distribution of PA in single cells as well as among different cells.Like that of other phospholipids, the regulation of phosphatidic acid (PA) 3 homeostasis is not simple, in part because PA can be a precursor or product of a number of metabolic pathways, and in part because several enzymes are involved in the production of PA. Two major precursors of PA are phosphatidylcholine and diacylglycerol (DAG), which are the substrates of phospholipase D (PLD) and DAG kinases, respectively (1). The produced PA is then dephosphorylated by PA phosphatases to yield DAG, or hydrolyzed by phospholipase A to yield lyso-PA. In mammalian cells, there are two major PLDs, PLD1 and PLD2, which are distinct from each other in terms of both overexpression and knock-down phenotype (2). In addition, PLD1 and PLD2 differ strikingly in subcellular localization (3). Such observations have suggested that the PA generation in different subcellular membrane compartments is regulated by different PLDs and that the PA in different subcellular compartment regulates different cellular functions. Similarly, mammalian DAG kinases comprise an extended family with 10 members classified into five different subtypes with different regulatory domains (4).Biochemical methods such as the radioisotopic labeling of phosphate or fatty acids have been used to measure cellular PA content. However, spatial information cannot be obtained though this approach. To overcome this problem, the yeast t-SNARE Spo20 (5), mammalian Raf1 kinase (6), the Rac1 exchang...

“…Phosphorylation of tyrosine residues on signaling molecules plays a pivotal role in a wide range of eukaryotic cellular processes (33,34), which also includes phosphorylation/activation of PLD by epidermal growth factor receptor (35,36) or by protein kinase C␣ (PKC␣) (37). We observed the presence of the tyrosine kinases Fes and JAK3 in MCF10A (as a model of an untransformed cell line) and MDA-MB-231 (as a model of transformed cells) and determined how these kinases regulate one another and also how they regulate PLD enzymatic activity.…”

Section: Discussionmentioning

confidence: 99%

A New Signaling Pathway (JAK-Fes-phospholipase D) That Is Enhanced in Highly Proliferative Breast Cancer Cells

Kantonen

Henkels

et al. 2013

Background: Phospholipase D is a signaling protein whose expression is elevated in transformed cells. Results:The tyrosine kinases JAK and Fes are responsible for high PLD activity and expression and high cell proliferation. Conclusion: A new signaling pathway (JAK-Fes-PLD) has been found whose operation is heightened in cancer cells. Significance: Modulating this new pathway could control the highly invasive phenotype of transformed cells.