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EVOLUTIONARY HOMOLOGS part 3/3

PLC gamma membrane targeting by interaction with inositol phospholipids

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. The inositol phospholipid known as phosphatidylinositol 3,4, 5-trisphosphate (PtdIns-3,4,5-P3) has been shown to bind to the C-terminal SH2 domain of phospholipase Cgamma (PLCgamma) with an apparent Kd of 2.4 microM and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivo relevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCgamma with or without receptor association with PI3K. Coactivation of PLCgamma and PI3K result in an approximately 40% increase in both intracellular IP3 generation and intracellular calcium release, as compared with selective activation of PLCgamma. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibits the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3 production and intracellular calcium release, potentially via enhanced PtdIns-4, 5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCgamma to the lipid bilayer (Rameh, 1998).

Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase C gamma (PLC gamma) and phosphatidylinositol (PI) 3-kinase. While it is well established that PLC gamma activation requires tyrosine phosphorylation, additional regulation of PLC gamma is provided by the lipid products of PI 3-kinase. The pleckstrin homology (PH) domain of PLC gamma binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLC gamma PH domain-mediated membrane targeting and PLC gamma activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLC gamma membrane targeting by overexpression of the PLC gamma PH domain prevents growth factor-induced PLC gamma activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism (Falasca, 1998).

Phospholipase C gamma signaling and downstream functions

Platelet-derived growth factor (PDGF) induces the phosphorylation of phospholipase C (PLC) gamma1. Phosphorylation on tyrosine (Tyr) 783 of PLCgamma1 is essential for phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vivo, while phosphorylation does not affect the catalytic activity in vitro. To study the roles of Tyr-783 phosphorylation in vivo, a polyclonal antibody containing phosphotyrosine 783 (alpha-PLCgamma1 PY) was developed that recognizes PLCgamma1 . Tyr-783-phosphorylated PLCgamma1 is not detected in the absence of PDGF:it appears after stimulation, increases for 30 min, and then decreases to near the prestimulation level. Immunostaining of cells show that PDGF-produced Tyr-783-phosphorylated PLCgamma1 localizes predominantly at membrane ruffles and stress fibers, where it colocalizes with actin filaments within 30 min. Ninety minutes after PDGF stimulation, the actin filaments are disassembled to short fragments, and the levels of Tyr-783-phosphorylated PLCgamma1 are remarkably decreased in membrane ruffles and cytoskeleton. Furthermore, the depolymerization of actin filaments and membrane ruffling caused by PDGF stimulation are blocked by microinjecting alpha-PLCgamma1 PY, such as occurs following the microinjection of the PLCgamma1-2SH2 domain, which is expected to associate with phosphorylated PDGF receptors and to block PLCgamma1 binding. It is worth noting that the microinjection of tyrosine-phosphorylated peptide (consisting of 13 amino acids containing Tyr-783) induces the disassembly of actin filaments and membrane ruffling as observed in PDGF-stimulated cells, while nonphosphorylated peptide does not cause any effect. These data suggest that the phosphorylation of PLCgamma1 on tyrosine 783 by PDGF plays an important role in cytoskeletal reorganization in addition to mitogenesis (Yu, 1998).

Phospholipase C gamma1 (PLC-gamma1) is phosphorylated upon treatment of cells with nerve growth factor (NGF). To assess the role of PLC-gamma1 in mediating the neuronal differentiation induced by NGF treatment, PC12 cells were established that overexpress whole PLC-gamma (PLC-gamma1PC12); the SH2-SH2-SH3 domain (PLC-gamma1SH223PC12); SH2-SH2-deleted mutants (PLC-gamma1deltaSH22PC12), and SH3-deleted mutants (PLC-gamma1deltaSH3PC12). Overexpressed whole PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 stimulates cell growth and inhibits NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-gamma1 that lack the SH2-SH2 domain or the SH3 domain have no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-gamma1 results in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-gamma1 does not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-gamma1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-gamma1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-gamma1 as well as intact PLC-gamma1 is tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-gamma1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-gamma1 but to the SH2-SH2-SH3 domain of PLC-gamma1 (Bae, 1998).

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. Evidence is presented for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma are observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, a specific inhibitor of PLC, U73122, was used to block insulin-stimulated GLUT4 translocation. Microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma blocks insulin-stimulated GLUT4 translocation. Inhibition of 2-deoxyglucose uptake is demonstrated in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. It is concluded that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes (Kayali, 1998).

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways; release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM; maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacities to release Ca2+ when a subsequent stimulus results in production of InsP3 (Wilson, 1998).

The cytoplasmic regions of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) bind and activate phospholipase C-gamma1 (PLC-gamma1) and other signaling proteins in response to ligand binding outside the cell. Receptor binding by PLC-gamma1 is a function of its SH2 domains and is required for growth factor-induced cell cycle progression into the S phase. Microinjection into MDCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide corresponding to the noncatalytic SH2-SH2-SH3 domains of PLC-gamma1 (PLC-gamma1 SH2-SH2-SH3) block growth factor-induced S-phase entry. Treatment of cells with diacylglycerol (DAG) alone, or just DAG and microinjected inositol-1,4,5-triphosphate (IP3), both products of activated PLC-gamma1, is not sufficient to stimulate cellular DNA synthesis, although such treatment does suppress the inhibitory effects of the PLC-gamma1 SH2-SH2-SH3 polypeptide (but not the cell cycle block imposed by inhibition of the adapter protein Grb2 or p21 Ras). Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, as well as a wild-type version (wtSRE-CAT) and a mutant (pm18) were used to investigate the function of PLC-gamma1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to both protein kinase C (PKC)-dependent and -independent signals, while the mutant, pm18, responds only to PKC-independent signals. Microinjection of the dominant-negative PLC-gamma1 SH2-SH2-SH3 polypeptide greatly reduces the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but has no effect on the responses of mutant pm18. These results indicate that in addition to the Grb2-mediated activation of Ras, PLC-gamma1-mediated DAG production is required for EGF- and PDGF-induced S-phase entry and gene expression, possibly through activation of PKC (Wang, 1998).

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. The FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, advantage was taken of this difference: receptor chimeras were prepared containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 have been identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction reveals that the JM region of FGFR-1 confers strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein is one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involves the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functions primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis reveals that the JM region does not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR (Lin, 1998).

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), targeted disruption of the Plcg1 gene in the mouse was used to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF show a time- and dose-dependent increase in the production of total inositol phosphates that is substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity, which has a similar dose dependence to the PLC response but is down-regulated after 15 min. Phospholipase D activity, however, is markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remain in the Plcg1(-/-) cells can be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells is phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells show Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells is activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells is not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin (to mimic the effect of PLC-gamma1) PLD activity is restored. The targeted disruption of Plcg1 does not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase is similar in Plcg1(+/+) and Plcg1(-/-) cells. Because an increase in plasma membrane ruffles occurs in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible that neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs (Hess, 1998).

Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. The phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, it is shown that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs (Rong, 2004).

Mutation of Phospholipase C gamma

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E 9.0. These results clearly demonstrate that PLC-gamma1 with (by inference) its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes (Ji, 1997).

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-gamma1, a ubiquitous tyrosine kinase substrate. Plcg1(-/-) embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1(+/+) embryos. The null cells do grow to a higher saturation density in serum-containing media, since their capacity to spread out is decreased when compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1(-/-) cells respond equivalently to PLcg1(+/+) cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-gamma1 for many responses to growth factors (Ji, 1998).

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