Three families of phospholipase C (PI-PLC, ␥, and ␦) are known to catalyze the hydrolysis of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, leading to a cascade of intracellular responses that result in cell growth, cell differentiation, and gene expression. Here we describe the founding member of a novel, structurally distinct fourth family of PI-PLC. PLC⑀ not only contains conserved catalytic (X and Y) and regulatory domains (C2) common to other eukaryotic PLCs, but also contains two Ras-associating (RA) domains and a Ras guanine nucleotide exchange factor (RasGEF) motif. PLC⑀ hydrolyzes PIP 2 , and this activity is stimulated selectively by a constitutively active form of the heterotrimeric G protein G␣ 12 . PLC⑀ and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTPRas. Thus PLC⑀ is a RasGEF. PLC⑀, the mutant H1144L, and the isolated GEF domain activate the mitogen-activated protein kinase pathway in a manner dependent on Ras but independent of PIP 2 hydrolysis. Our findings demonstrate that PLC⑀ is a novel bifunctional enzyme that is regulated by the heterotrimeric G protein G␣ 12 and activates the small G protein Ras/mitogen-activated protein kinase signaling pathway.Many hormones, neurotransmitters, and growth factors elicit intracellular responses by activating a family of inositol phospholipid-specific phospholipase C (PLC) 1 isozymes (1, 2).There are three established families of PLC termed  (ϳ150 kDa), ␥ (ϳ145 kDa), and ␦ (ϳ 85 kDa) (3). All three families of the PI-PLC family are able to recognize phosphatidylinositol (PI), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and to carry out the Ca 2ϩ -dependent hydrolysis of these inositol phospholipids. It is presumed that the primary substrate for hydrolysis is PIP 2 , which yields the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 releases intracellular Ca 2ϩ from the endoplasmic reticulum via interaction with a specific receptor located on the surface of the endoplasmic reticulum. DAG, as well as increased intracellular Ca 2ϩ , activate protein kinase C leading to a cascade of intracellular events including regulation of cellular growth, smooth muscle contraction, and cardiac hypertrophy (2).The mode of regulation differs considerably for members of the different isoform families. The  isoforms are regulated by large heterotrimeric G proteins. After activation by agonists such as epinephrine, ␣ 1 adrenergic receptors are able to couple to G␣ subunits from the G q /G 11 family and stimulate hydrolysis of phosphatidyl inositol lipids via PLC  isoforms. For some  isoforms, G q alone is sufficient for activation, whereas for others the coordinated action of both G q and ␥ is necessary. The ␥ isoforms of PLC contain SH2 and SH3 domains; hence, they are activated by both receptor and nonreceptor tyrosine kinases. Until recently, ...
The deduced amino acid sequence shows 53 and 90% identity to hPLD1 and rodent PLD2, respectively. The mRNA for PLD2 was widely distributed in various tissues including peripheral blood leukocytes, and the distribution was distinctly different from that of hPLD1. hPLD1 and hPLD2 both showed a requirement for phosphatidylinositol 4,5-bisphosphate. Both isoforms showed optimal activity at 10-20 mol % phosphatidylcholine in a mixed lipid vesicle system and showed comparable basal activities in the presence of phosphatidylinositol 4,5-bisphosphate. Unexpectedly, ARF-1 stimulated the activity of hPLD2 expressed in insect cells about 2-fold, compared with a 20-fold stimulation of hPLD1 activity. Thus, not only PLD1 but also hPLD2 activity can be positively regulated by both phosphatidylinositol 4,5-bisphosphate and ARF. Phospholipase D (PLD)1 has been implicated in a wide range of physiological processes and diseases including inflammation, secretion, mitogenesis, neuronal and cardiac stimulation, diabetes, and the respiratory burst in neutrophils (1). PLD catalyzes the hydrolysis of phospholipids, usually phosphatidylcholine, to generate phosphatidic acid plus the head group. Phosphatidic acid may act directly as a signaling molecule (2-5) or can be further metabolized to form diacylglycerol by phosphatidic acid phosphohydrolase (6). The latter may function as an activator of protein kinase C isoforms and possibly other diacylglycerol-dependent enzymes. PLD can be activated in cells by a variety of extracellular agonists including those that bind to G protein-coupled receptors (e.g. the fMet-Leu-Phe receptor in neutrophils) and those that stimulate receptor tyrosine kinases (3,7,8). Phorbol esters are also potent activators of PLD in a variety of cell systems, implicating protein kinase C in PLD regulation. PLD activities from mammalian cells were initially studied in broken or permeabilized cell preparations. ADP-ribosylation factor (ARF) was shown to be a potent activator in rat brain preparations (9) and in permeabilized HL-60 cells (10). PLD activity found in the cytosolic fraction obtained from HL-60 was also activated by ARF (5). A role for Rho family small GTPases such as RhoA, Rac, and Cdc42 was established based on inhibition by RhoGDI and by reconstitution studies (11-13). PLD activity in plasma membranes from rat liver was stimulated by RhoA but not ARF (13). In some systems, RhoA and ARF synergize to increase PLD activity (5, 14 -16). The actions of RhoA and ARF can be further enhanced by cytosolic factors (12, 16 -19) as well as by protein kinase C (5,15,16,20). Protein kinase C activates PLD in a phosphorylation-dependent (21) or phosphorylation-independent (16, 22) manner, depending on the system. Solubilized preparations required PIP 2 for activity (9). These and other differences (e.g. subcellular localization, Ca 2ϩ requirement, activation or inhibition by oleate, etc.) implied that several isoforms of PLD exist (3, 23).The first cloned mammalian homologue of a plant PLD, human PLD1 (hPLD1), is reg...
Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate-stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 W WM in vitro. Intriguingly, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) synergistically stimulates the oleate-dependent PLD2 activity with an optimal concentration of 2.5 W WM. These results provide the first evidence that oleate is a PLD2-specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP 2 .z 1999 Federation of European Biochemical Societies.
In a variety of intact cells, phorbol esters are known to activate phospholipase D. In a cell-free system consisting of plasma membrane and cytosol from human neutrophils, phorbol esters activated phospholipase D in an adenosine nucleotide triphosphate-dependent manner. ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was 2-3-fold more effective than ATP, while ADP and AppNHp (adenyl-5'-yl imidodiphosphate) were ineffective, and activation was blocked by the kinase inhibitor staurosporine. In cytosol deplete of protein kinase C by chromatography on threnoine-Sepharose, phorbol ester-dependent activation was lost, but was restored upon addition of purified rat brain protein kinase C. The target for phosphorylation was shown to be the plasma membrane plasma membrane was phosphorylated using ATP gamma S/phorbol 12,13-dibutyrate and protein kinase C and was reisolated to remove activators. Upon adding nucleotide-depleted cytosol, activator-independent phospholipase D activity was seen. Using this prephosphorylation protocol, PKC-dependent activation of plasma membranes was found to require micromolar calcium, implicating a conventional protein kinase C. Using recombinant isoforms of protein kinase C, only the conventional isoforms showed significant activation, with the following rank order of potency: beta 1 > alpha > gamma; the beta 2, delta, epsilon, eta, and sigma isoforms showed little or no activity. Thus, conventional isoform(s) of protein kinase C activate neutrophil phospholipase D by phosphorylating a target protein located in the plasma membrane.
Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association.
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