In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC−γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC−γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC−γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC−γ1 activation are regulated both independently and additively by recruitment of PLC−γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC−γ1 tetramer, and by tyrosine phosphorylation of PLC−γ1. The recently solved structure of PLC−γ1 indicates that, in the resting state, several PLC−γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC−γ1, enabling contact with its membrane substrate, disinhibition of PLC−γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.     

A naturally occurring membrane-anchored Gαs variant,XLαs,activates phospholipase Cβ4

Extra-large stimulatory Gα (XLα_s) is a large variant of G protein α_s subunit (Gα_s) that uses an alternative promoter and thus differs from Gα_s at the first exon. XLα_s activation by G protein–coupled receptors mediates cAMP generation, similarly to Gα_s; however, Gα_s and XLα_s have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLα_s can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLα_s directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLα_s to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLα_s relative to Gα_s confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of G_s-coupled receptors that may have a critical role in endocrine physiology.     

Epidermal growth factor and platelet-derived growth factor promote translocation of phospholipase C-γ from cytosol to membrane

Treatment of HER 14 cells with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) induced a translocation of phospholipase C-γ (PLC-γ) from cytosol to membrane. In such growth factor-treated cells, cytosolic PLC-γ was found to contain more phosphotyrosine than membrane-associated enzyme. Because these growth factors have been shown to promote both the physical association of PLC-γ with their receptors and the subsequent phosphorylation of the enzyme directly by the membrane-bound receptor tyrosine kinases, the membrane assocation of PLC-γ may simply be due to the formation of transient enzyme (receptor)-substrate (PLC-γ) complexes. If this is the case, membrane-associated PLC-γ would be expected to be released from membrane after undergoing tyrosine phosphorylation. However, tyrosine phosphorylation of membrane-associated PLC-γ by the EGF receptor in vitro did not result in the release of PLC-γ from membrane. Thus, the association of PLC-γ with membrane would appear to involve more than enzyme-substrate complex.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Identification of phospholipase C gamma1 as a protein tyrosine phosphatase mu substrate that regulates cell migration

The receptor protein tyrosine phosphatase PTPµ has a cell‐adhesion molecule‐like extracellular segment and a catalytically active intracellular segment. This structure gives PTPµ the ability to transduce signals in response to cell–cell adhesion. Full‐length PTPµ is down‐regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPµ may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPµ substrates in tumor cell lines. We identified both PKCδ and PLCγ1 as PTPµ substrates. As PLCγ1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPµ may be upstream of PLCγ1 in regulating glioma cell migration. We conducted brain slice assays using U87‐MG human glioma cells in which PTPµ expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPµ shRNA and a PLCγ1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCγ1 is downstream of PTPµ and that dephosphorylation of PLCγ1 is likely to be a major pathway through which PTPµ suppresses glioma cell migration. J. Cell. Biochem. 112: 39–48, 2011. © 2010 Wiley‐Liss, Inc.     

PLCγ1 Participates in Protein Transport and Diacylglycerol Production Triggered by cargo Arrival at the Golgi

Diacylglycerol (DAG) is required for membrane traffic and structural organization at the Golgi. DAG is a lipid metabolite of several enzymatic reactions present at this organelle, but the mechanisms by which they are regulated are still unknown. Here, we show that cargo arrival at the Golgi increases the recruitment of the DAG‐sensing constructs C1‐PKCθ‐GFP and the PKD‐wt‐GFP. The recruitment of both constructs was reduced by PLCγ1 silencing. Post‐Golgi trafficking of transmembrane and soluble proteins was impaired in PLCγ1‐silenced cells. Under basal conditions, PLCγ1 contributed to the maintenance of the pool of DAG associated with the Golgi and to the structural organization of the organelle. Finally, we show that cytosolic phospholipase C (PLC) can hydrolyse phosphatidylinositol 4‐phosphate in isolated Golgi membranes. Our results indicate that PLCγ1 is part of the molecular mechanism that couples cargo arrival at the Golgi with DAG production to co‐ordinate the formation of transport carriers for post‐Golgi traffic.      

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [³²P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca²⁺ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC and , the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [³²P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.     

Mechanistic insights on novel small molecule allosteric activators of cGMP-dependent protein kinase PKG1α

cGMP-dependent protein kinase (PKG) represents a compelling drug target for treatment of cardiovascular diseases. PKG1 is the major effector of beneficial cGMP signaling which is involved in smooth muscle relaxation and vascular tone, inhibition of platelet aggregation and signaling that leads to cardioprotection. In this study, a novel piperidine series of activators previously identified from an ultrahigh-throughput screen were validated to directly bind partially activated PKG1α and subsequently enhance its kinase activity in a concentration-dependent manner. Compounds from initial optimization efforts showed an ability to activate PKG1α independent of the endogenous activator, cGMP. We demonstrate these small molecule activators mimic the effect of cGMP on the kinetic parameters of PKG1α by positively modulating the K_M of the peptide substrate and negatively modulating the apparent K_M for ATP with increase in catalytic efficiency, k_cat. In addition, these compounds also allosterically modulate the binding affinity of cGMP for PKG1α by increasing the affinity of cGMP for the high-affinity binding site (CNB-A) and decreasing the affinity of cGMP for the low-affinity binding site (CNB-B). We show the mode of action of these activators involves binding to an allosteric site within the regulatory domain, near the CNB-B binding site. To the best of our knowledge, these are the first reported non-cGMP mimetic small molecules shown to directly activate PKG1α. Insights into the mechanism of action of these compounds will enable future development of cardioprotective compounds that function through novel modes of action for the treatment of cardiovascular diseases.     

Identification of Gαi3 as a promising target for osteosarcoma treatment

Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression. In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression.     

Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts

The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F₄⁻, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F₄⁻ -promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4–5-fold increase in the formation of [³H]inositol-1,4-phosphate and [³H]inositol-1,4,5-phosphate; and a 36% increase in [³H]inositol-1-phosphate within 5 min in prelabeled cells. [³H]inositol-4-phosphate, a metabolite of [³H]inositol-1,4-phosphate and [³H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F₄⁻ -activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [³H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F₄⁻ by 90–100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F₄⁻-induced [³H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C. J. Cell. Biochem. 65:198–208. © 1997 Wiley-Liss, Inc.     

Phosphorylation of Phospholipase C‐δ1 Regulates its Enzymatic Activity

Phosphorylation of phospholipase C‐δ₁ (PLC‐δ₁) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC‐δ₁ most potently. It was also demonstrated that PLC‐δ₁ directly bound PKC‐α via its pleckstrin homology (PH) domain. Using deletion mutants of PLC‐δ₁ and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC‐δ₁. In vitro phosphorylation of PLC‐δ₁ by PKC stimulated [³H]PtdIns(4,5)P₂ hydrolyzing activity and [³H]Ins(1,4,5)P₃‐binding of the PLC‐δ₁. On the other hand, endogenous PLC‐δ₁ was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, we determined that Thr209 of PLC‐δ₁ is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC‐δ₁ was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC‐α reduced serine phosphorylation of PLC‐δ₁ detected by an anti‐phosphoserine antibody and PLC‐δ₁‐dependent basal production of inositol phosphates in NIH‐3T3 cells, suggesting PKC‐α activates phosphatase or inactivates another kinase involved in PLC‐δ₁ serine phosphorylation to modulate the PLC‐δ₁ activity in vivo. Taken together, these results suggest that PLC‐δ₁ has multiple phosphorylation sites and phosphorylation status of PLC‐δ₁ regulates its activity positively or negatively depends on the phosphorylation sites. J. Cell. Biochem. 108: 638–650, 2009. © 2009 Wiley‐Liss, Inc.     

Protein kinase C activation by interleukin (IL)-1 limits IL-1-induced IL-6 synthesis in osteoblast-like cells: Involvement of phosphatidylcholine-specific phospholipase C

We investigated the regulatory mechanism of interleukin-6 (IL-6) synthesis induced by interleukin-1 (IL-1) in osteoblast-like MC3T3-E1 cells. IL-1 stimulated the secretion of IL-6 in a dose-dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL-1-induced secretion of IL-6. The stimulative effect of IL-1 was markedly amplified in PKC down-regulated MC3T3-E1 cells. IL-1 produced diacylglycerol in MC3T3-E1 cells. IL-1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL-1 significantly stimulated the formation of phosphocholine dose-dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the IL-1-induced diacylglycerol production. The IL-1-induced IL-6 secretion was significantly enhanced by D-609. These results indicate that IL-1 activates PKC via phosphatidylcholine-specific phospholipase C in osteoblast-like cells, and the PKC activation then limits IL-6 synthesis induced by IL-1 itself. J. Cell. Biochem. 67:103–111, 1997. © 1997 Wiley-Liss, Inc.     

The molecular mechanism of acylation stimulating protein regulation of adipophilin and perilipin expression: involvement of phosphoinositide 3-kinase and phospholipase C

The novel adipokine acylation stimulating protein (ASP) is involved in lipid metabolism and obesity‐related disorders. Adipophilin and perilipin, two members of the lipid droplet protein family, participate not only in fat storage within adipocytes, but also in ectopic lipid deposition in the form of cytoplasmic triglyceride (TG) droplets within many types of mammalian cells. During differentiation to mature adipocytes, mechanisms controlling the synthesis and turnover of these lipid droplet proteins are only partially understood, the mechanisms regulating gene/protein expression as yet unidentified. In our previous study, ASP has been shown to regulate adipophilin and perilipin expression to facilitate TG synthesis during 3T3‐L1 cell differentiation. Our aim in this study was to provide insight into the physiological importance of phosphoinositide 3‐kinase (PI3K) and phospholipase C (PLC) in ASP‐triggered alteration of adipophilin and perilipin expression. We found that acute (2.5 h) inhibition of PLC or PI3K results in a decrease in mRNA and protein of perilipin and adipophilin at any time during differentiation. The fact that there is such a rapid change even with mRNA levels suggests a rapid turnover of both mRNA and protein independent of a direct ASP effect. Also, the presence of these inhibitors blocked the ASP stimulatory effects with a maximal decrease in gene and protein expression of adipophilin (?45% and ?60%, respectively, P < 0.01) and perilipin (?96% and ?63%, respectively, P < 0.01 and P < 0.05). These findings provide further understanding of the adipogenic properties of ASP in adipocytes. J. Cell. Biochem. 112: 1622–1629, 2011. © 2011 Wiley‐Liss, Inc.     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.     

Involvement of phosphatidylcholine-selective phospholipase C in activation of mitogen-activated protein kinase pathways in imidazoline receptor antisera-selected protein

Imidazoline receptor antisera-selected protein (IRAS) is considered as a candidate for the I1-imidazoline receptor (I1R), but the signaling pathway mediated by IRAS remains unknown. In our study, the signal transduction pathways of IRAS were investigated in CHO cells stably expressing IRAS (CHO-IRAS), and compared to the native I1R signaling pathways. Rilmenidine or moxonidine (10 nM-100 microM), I1R agonists, failed to stimulate [35S]-GTPgammaS binding in CHO-IRAS cell membrane preparations, suggesting that G protein may not be involved in IRAS signaling pathway. However, incubation of CHO-IRAS with rilmenidine or moxonidine for 5 min could induce an upregulation of phosphatidylcholine-selective phospholipase C (PC-PLC) activity, and an increase in the accumulation of diacylglycerol (DAG), the hydrolysate of PC-PLC, in a concentration-dependent manner. The elevated activation of PC-PLC by rilmenidine or moxonidine (100 nM) could be blocked by efaroxan, a selective I1R antagonist. Cells treated with rilmenidine or moxonidine showed an increased level of extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, which could be reversed by efaroxan or D609, a selective PC-PLC inhibitor. These results suggest that the signaling pathway of IRAS in response to I1R agonists coupled with the activation of PC-PLC and its downstream signal transduction molecule, ERK. These findings are similar to those in the signaling pathways of native I1R, providing some new evidence for the relationship between I1R and IRAS.     

The role of PLCγ2 in immunological disorders,cancer, and neurodegeneration

Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) is a critical signaling molecule activated downstream from a variety of cell surface receptors that contain an intracellular immunoreceptor tyrosine-based activation motif. These receptors recruit kinases such as Syk, BTK, and BLNK to phosphorylate and activate PLCγ2, which then generates 1D-myo-inositol 1,4,5-trisphosphate and diacylglycerol. These well-known second messengers are required for diverse membrane functionality including cellular proliferation, endocytosis, and calcium flux. As a result, PLCγ2 dysfunction is associated with a variety of diseases including cancer, neurodegeneration, and immune disorders. The diverse pathologies associated with PLCγ2 are exemplified by distinct genetic variants. Inherited mutations at this locus cause PLCγ2-associated antibody deficiency and immune dysregulation, in some cases with autoinflammation. Acquired mutations at this locus, which often arise as a result of BTK inhibition to treat chronic lymphocytic leukemia, result in constitutive downstream signaling and lymphocyte proliferation. Finally, a third group of PLCγ2 variants actually has a protective effect in a variety of neurodegenerative disorders, presumably by increased uptake and degradation of deleterious neurological aggregates. Therefore, manipulating PLCγ2 activity either up or down could have therapeutic benefit; however, we require a better understanding of the signaling pathways propagated by these variants before such clinical utility can be realized. Here, we review the signaling roles of PLCγ2 in hematopoietic cells to help understand the effect of mutations driving immune disorders and cancer and extrapolate from this to roles which may relate to protection against neurodegeneration.