Activation of the enzyme PLC (phospholipase C) leads to the formation of second messengers Ins(1,4,5)P3 and diacylglycerol. RTKs (receptor tyrosine kinases) activate this reaction through PLCγ isoenzymes. It has been shown that PI3K (phosphoinositide 3-kinase) may regulate PLCγ activity through the interaction of PI3K product PtdIns(3,4,5)P3 and the PLCγ PH domain (pleckstrin homology domain). Here, we analyse the potential functional roles of the PI3K/PLC pathway.
- growth factor
- phospholipase C (PLC)
- pleckstrin homology domain (PH domain)
- phosphoinositide 3-kinase (PI3K)
- Src homology 2 domain (SH2 domain)
- tyrosine phosphorylation
PtdIns(4,5)P2 is a minor phospholipid component of cell membrane (∼0.5% of total lipids) that is synthesized primarily by the type I phosphatidylinositol-4-phosphate 5-kinase from PtdIns4P. PtdIns(4,5)P2 is specifically enriched at the plasma membrane where it is an important substrate for several signalling proteins. For instance PLC (phospholipase C) hydrolyses the phosphodiester link in PtdIns(4,5)P2 leading to the formation of second messengers Ins(1,4,5)P3 and diacylglycerol. PtdIns(4,5)P2 is also a substrate for class I PI3Ks (phosphoinositide 3-kinases), the enzymes that phosphorylate the position 3 of the inositol ring of PtdIns(4,5)P2 generating PtdIns(3,4,5)P3.
PtdIns(3,4,5)P3 possesses second messenger properties via interaction with specific protein modular domains such as the PH domain (pleckstrin homology domain) . This lipid–protein interaction is involved in membrane targeting of proteins to specific cellular compartments. The PH domain is characterized by a low sequence homology but a high structural identity. Initially identified as a protein-binding domain, the PH domains were afterwards shown to be able to interact with phosphoinositides. In particular, PtdIns(3,4,5)P3 was found to bind the PH domain of different proteins among which the PKB (protein kinase B)/Akt is the best characterized. The mechanism of Akt activation is generally considered paradigmatic of PI3K/PH domain-mediated activation and this involves the growth factor- or cytokines-dependent PI3K activation with the subsequent generation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2. A direct consequence of their synthesis on the plasma membrane is the recruitment of Akt, present in an inactivated form in the cytosol, and activated to the plasma membrane.
Although Akt is certainly the best-characterized downstream target of PI3K, many other proteins have been proposed as downstream PI3K targets, including PLCγ1 .
PLCγ has been involved in different processes including cell proliferation and cell motility. The main mechanism of activation is via receptor RTK (receptor tyrosine kinase) tyrosine phosphorylation and recruitment to the receptor through its two SH2 domains (Src homology 2 domains) . Nevertheless, tyrosine phosphorylation is not sufficient for full activation of PLCγ1 in cells [3,4]. Additional mechanisms of activation have been found that can be complementary or required for full activation of PLCγ upon RTK stimulation. In fact, previous evidence indicates that PLCγ isoenzymes are additionally activated by phosphatidic acid, PtdIns(3,4,5)P3 and arachidonic acid in the absence of PLCγ tyrosine phosphorylation .
Two different protein domains of PLCγ1 have been proposed to be PtdIns(3,4,5)P3-binding domains, the PH domain and the SH2 domain [2,5]. The N-terminal PH domain of PLCγ interacts with lipid product PtdIns(3,4,5)P3  and might provide a means to localize PLCγ1 to the plasma membrane. Although yeast two-hybrid analysis did not support a role for the interaction of PLCγ1 N-terminal PH domain with PtdIns(3,4,5)P3 , molecular modelling  and more recent evidences confirm that the domain is crucial for PLCγ1 membrane association and PI3K-dependent activation . In particular, mutation of the amino acid residues responsible for PtdIns(3,4,5)P3 binding in the intact PLCγ1 molecule impairs membrane targeting . In addition, these results demonstrate that the PH domain is not required for the initial EGF (epidermal growth factor)-induced translocation of PLCγ1 from the cytosol to the plasma membrane, but is involved in a second step of PLCγ1 activation .
Different signalling pathways involve a PI3K-dependent regulation of PLCγ, such as those activated by T-cell receptor, insulin receptor and vitamin D3. Interestingly, the expression of the tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10), in human glioblastoma cells reduces the intracellular level of Ins(1,4,5)P3, suggesting that PTEN inhibits PLC activity , possibly through inhibition of a PI3K-dependent pathway.
PI3K-dependent activation of PLCγ2 has been reported in B-cells, macrophages and platelets . PI3K and PLCγ have both been implicated as critical mediators of B-cell activation and differentiation signals. Previous results indicate that PLCγ2 activation is dependent on PtdIns(3,4,5)P3 formation and that these two pathways are interconnected at several levels [3,10,11] but the precise mechanism of PLCγ2 regulation is not clear. Cytoplasmic protein tyrosine kinases, such as Btk (Bruton's tyrosine kinase), play a critical role in activation of PLCγ2 and PI3K. Interestingly, Btk activation is also mediated by interaction of its PH domain with PtdIns(3,4,5)P3 and consequential membrane recruitment. Current models therefore propose a dual PI3K-dependent regulation of PLCγ2, mediated by the interaction between Btk and PLCγ2 PH domains and PtdIns(3,4,5)P3 [10,11].
These results indicate that PI3K is necessary for PLCγ activation, although at present it is not clear whether PtdIns(3,4,5)P3 alone is also sufficient to activate the enzyme in the absence of tyrosine phosphorylation. PtdIns(3,4,5)P3 induces strong activation of PLCγ1 in vitro , whereas constitutively active PI3K is able to induce PLCγ1 activation in vivo . Interestingly, it has been shown that during Listeria monocytogenes invasion, activation of PLCγ1 occurs without tyrosine phosphorylation in a mechanism completely dependent on PI3K activation . In contrast, in HepG2 cells, PDGF (platelet-derived growth factor)-induced activation of PI3K is not sufficient to induce PLCγ1 activation and tyrosine phosphorylation is a prerequisite . Therefore these results suggest that different growth factor receptors or context may activate PLCγ1 in different ways.
In an effort to understand the physiological roles of the PI3K-dependent PLCγ1, we have previously found that EGF induces a PI3K-dependent translocation of PLCγ1 at the leading edge of migrating breast cancer cells . Our experiments also showed that stable PLCγ1 PH domain expression blocks EGF-induced cell motility , indicating that a PI3K/PLCγ1 pathway is important in cell migration.
In addition it has been reported that a PI3K-dependent PLCγ activation may play a crucial role in Escherichia coli K-1 interaction with human brain microvascular endothelial cells .
Furthermore, previous results suggest that a PI3K/PLCγ1 pathway might play a key role in angiogenesis. Absence of vasculogenesis has been reported in PLCγ1-deficient mice . Previously, a mutant (y10) that displays a specific defect in the formation of arteries, but not veins, has been identified in zebra fish . Interestingly, y10 encodes the zebra fish homologue of PLCγ1 and the y10 mutants fail to respond to exogenous VEGF (vascular endothelial growth factor), indicating that PLCγ1 functions specifically downstream of the VEGF receptor. In addition, a striking similarity has been found between the defects found in the zebra fish PLCγ1 y10 mutant phenotype and embryos lacking VEGF. Interestingly, sequence analysis revealed that most of the defects in y10 mutants occur in the PH domain . More specifically, amino acids mutations are located in the β3/β4 region of the PH domain that we demonstrated to be important for PtdIns(3,4,5)P3 binding and growth factor-induced membrane localization of PLCγ1 . These results suggest that a PI3K-dependent regulation of PLCγ1 might be involved in vasculogenesis.
Although it is clear that PtdIns(3,4,5)P3 is a crucial player in PLCγ activation, its role is often disregarded. Possibly, this is due to the fact that the mechanism underlying the PI3K-dependent regulation of PLCγ is not fully elucidated as for Akt activation. Nevertheless, a large body of evidence exists to support the role of PI3K in PLCγ activation. We hypothesize that the mechanisms by which PtdIns(3,4,5)P3 enhances PLCγ activation could be cell-type- and growth-factor-dependent.
Our work is supported by the British Heart Foundation, Diabetes UK, Association for International Cancer Research and Fondazione Carichieti.
3rd Focused Meeting on PI3K Signalling and Disease: Biochemical Society Focused Meeting held at Bath Assembly Rooms, U.K., 6–8 November 2006. Organized and Edited by B. Hemmings (Friedrich Miescher Institute for Biomedical Research, Switzerland), B. Vanhaesebroeck (Ludwig Institute for Cancer Research, U.K.), S. Ward (Bath, U.K.) and M. Welham (Bath, U.K.).
Abbreviations: Btk, Bruton's tyrosine kinase; EGF, epidermal growth factor; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RTK, receptor tyrosine kinase; SH2 domain, Src homology 2 domain; VEGF, vascular endothelial growth factor
- © 2007 The Biochemical Society