v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Abstract

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with araehidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG pkroduction and. enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.Protein kinase C (PKC) activity has been implicated in v-Src-induced intracellular signals (5,12,26,33,35,37,43). Diacylglycerol (DAG) is a second messenger that leads to the activation of PKC (25). DAG is generated from phospholipids either directly, through the action of type C phospholipases, or indirectly, through the action of type D phospholipases that generate phosphatidic acid (PA), which is then converted to DAG by a PA phosphatase (reviewed by Exton [10]). Increased levels of DAG have been observed in cells transformed by v-Src (22, 43); however, the source of the increased DAG is unclear. The time course for increased inositol phosphate levels in v-Src-transformed cells did not correlate with the time course for increased DAG (15,22), suggesting that DAG produced in response to v-Src is generated from a source other than phosphatidylinositol (P1) or phosphorylated derivatives of PI. Over the last several years, considerable data have been presented implicating PI hydrolysis by type C phospholipases as the source of increased DAG levels in response to a variety of stimuli (reviewed by Berridge [2]). Although it is likely that type C phospholipase-mediated hydrolysis of PI constitutes a major signaling pathway leading to the production of biologically active inositol phosphates, it is not clear that increases in DAG levels seen in response to a variety stimuli can be accounted for by hydrolysis of this relatively minor phospholipid (10). In this regard, it has recently been demonstrated that phosphatidylcholine (PC) rather than PI is the major source of increased DAG production in response to a variety of stimuli (la, 3, 6, 14, 19, 20, 23, 30, 32, 42 then washed with isotonic Tris-saline buffer, rapidly treated with 0.6 ml of methanol (MeOH)-6 N HCl (50:2), and scraped from the culture dish. The MeOH-HCl-treated cells were then extracted with 0.6 ml of CHCl3. Phase separation was obtained by adding 200 ,ul of 1 M NaCl. The organic phase was recovered, dried under N2, ...

Alternative phospholipase D/mTOR survival signal in human breast cancer cells

“…PLD activity has been reported to be elevated in cells transformed by v-Src (Song et al, 1991), v-Ras (Jiang et al, 1995a), and v-Raf . PLD activity is also elevated in response to mitogens including platelet-derived growth factor (Plevin et al, 1991), epidermal growth factor (Song et al, 1994), and insulin (Shome et al, 1997).…”

Activation of phospholipase D activity in transforming growth factor-beta-induced cell growth inhibition

“…Lipids were extracted and analyzed as previously described [15] with minor modifications. Reactions were stopped by quickly pouring off the medium and treating the cells with 600 l of ice-cold CH 3 OH/ 6 N HCl (50:2, v:v).…”

“…Many growth factors for receptor tyrosine kinases, including the epidermal growth factor (EGF), platelet-derived growth factor (PDGF), v-Src, fibroblast growth factor (FGF), and insulin-like growth factor (IGF), have been shown to activate PLD activity [9][10][11][12][13][14]. Activation of non-receptor tyrosine kinase v-Src has also been shown to activate PLD activity [15]. The involvement of protein kinase C in the regulation of PLD activity has been intensively studied in the past decade.…”

RalA requirement for v-Src- and v-Ras-induced tumorigenicity and overproduction of urokinase-type plasminogen activator: involvement of metalloproteases

“…Signaling pathways activated by v-Src and v-Ras (Burgering and Bos, 1995) include those involving phosphatidylinositol-3-kinase (Carpenter and Cantley, 1996), protein kinase C (Song et al, 1991), Raf-1 (Blobe et al, 1994) the extracellular regulated kinases (ERK-1 and 2) (Burgering and Bos, 1995) and phospholipase D (PLD) (Song et al, 1991). It has been reported that Raf-1, MEK-1 and ERK1 mediate the signal leading to uPA, uPA receptor, and MMP-9 upregulation Gum et al, 1997).…”