Activation of Phospholipase C-γ by Phosphatidylinositol 3,4,5-Trisphosphate

Abstract

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), a substrate for both enzymes, is converted to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P 3 activates purified PLC-␥ isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-␥ isozymes indirectly, in the absence of PLC-␥ tyrosine phosphorylation, through the generation of PI(3,4,5)P 3 .Activation of both PLC 1 and PI 3-kinase often occurs in response to stimulation of cells by a variety of agonists. PLC catalyzes the hydrolysis of PI(4,5)P 2 to generate the second messengers inositol 1,4,5-trisphosphate (I(1,4,5)P 3 ) and diacylglycerol (1-3). PI 3-kinase phosphorylates the D-3 position of PI(4,5)P 2 to produce PI(3,4,5)P 3 , which is then sequentially dephosphorylated to PI(3,4)P 2 and phosphatidylinositol 3-phosphate (4 -7). The activation of each of these two enzymes has been implicated in such diverse cellular processes as mitogenesis, chemotaxis, secretion, and cytoskeletal assembly (4 -7).The phosphoinositides PI(3,4)P 2 and PI(3,4,5)P 3 are not substrates of any known PLC (8) and are normally absent from resting cells; however, they appear within seconds to minutes of stimulation of cells with various growth factors or other cellular activators. In contrast, the concentration of phosphatidylinositol 3-phosphate does not change substantially in response to cell stimulation (4 -7). It has thus been suggested that PI(3,4)P 2 and PI(3,4,5)P 3 might function as intracellular messengers (4 -7). With regard to potential targets of these D-3-phosphorylated lipids, they have been shown to activate Ca 2ϩ -independent isoforms of protein kinase C (9, 10) as well as to bind the pleckstrin homology (PH) domain of the protein serine-threonine kinase Akt, thereby activating its kinase activity (11-13), and to the SH2 domains of the 85-kDa (p85) subunit of PI 3-kinase, thereby preventing its binding to tyrosine-phosphorylated proteins (14).The 10 mammalian PLC isozymes identified to date are single polypeptides and can be divided into three types: PLC-␤, PLC-␥, and PLC-␦ (1). All contain a PH domain in their NH 2 -terminal region. The ␥ type isozymes differ from the other two types in that they contain two SH2 domains, one SH3 domain, and an additional PH domain that is split by the SH domains; these domains are arranged in the order PH(N)-SH2-SH2-SH3-PH(C), where N and C in parentheses denote NH 2 -and COOHterminal locations, respectively. Upon st...

Protein tyrosine kinase‐dependent release of intracellular calcium in the sea urchin egg

“…Alternatively, the activation of PLC␥ is not directly dependent on tyrosine kinase activity (Rhee & Bae 1997). Phosphoinositide 3-kinase activity may activate PLC␥ by interaction at their SH2 domains in the absence of tyrosine phosphorylation (Bae et al 1998). There may also be the possibility of both PLC␤-and PLC␥-dependent InsP3 production controlling Ca 2+ release, as may be the case in mouse eggs during fertilization (Dupont et al 1996;Sette et al 1997;Williams et al 1998).…”

Fibroblast growth factor (FGF) signaling through PI 3-kinase and Akt/PKB is required for embryoid body differentiation

“…It has been demonstrated before, that PtdIns(4,5)-bis phosphate, phosphorylated at the 3' position by PI 3-kinase binds the PH domain of PLCg-1 and targets it to the cell membrane, where it binds FGFR1 (Bae et al, 1998;Falasca et al, 1998). A feedback of PtdIns(4,5)-bisphosphate then could activate PI 3-kinase.…”

“…Activation of PI 3-kinase is connected, through its lipid second messengers, to the activation of PLCg-1 (Bae et al, 1998;Falasca et al, 1998) and to the common auto phosphorylation site, Y766 of FGFR1 (Cross et al, 2000). To investigate the role of PLCg-1 in embryoid body di erentiation, mutant and wild type cultures were activated by heparin and FGF1, with or without the PI 3-kinase inhibitor, wortmannin.…”

The Recruitment of Phosphatidylinositol 3-Kinase to the E-cadherin-Catenin Complex at the Plasma Membrane Is Required for Calcium-induced Phospholipase C-γ1 Activation and Human Keratinocyte Differentiation

“…PI3K catalyzes the local production of PIP 3 (49), which activates PLC-␥1 (16,18). PI3K forms a complex with E-cadherin after calcium stimulation (24).…”

“…PLC-␥1 activation and human keratinocyte differentiation induced by calcium require phosphatidylinositol 3-kinase (PI3K) activation (16). PI3K converts PIP 2 to phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), which binds to the N-terminal PH domain (17) and the C-terminal SH 2 domain (18,19) of PLC-␥1 to activate PLC-␥1 (16). However, the mechanism by which calcium activates PI3K responsible for PLC-␥1-mediated human keratinocyte differentiation remains unclear.…”