Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

Background:  

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for successful completion of cytokinesis. In addition, both PIP₂ and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP₂ to yield diacylglycerol (DAG) and inositol trisphosphate (IP₃), which in turn induces calcium (Ca²⁺⁾ release from the ER. Several studies suggest PIP₂ must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP₂ regulates cytokinesis remains to be elucidated.     

Vitellogenesis in Oncopeltusfasciatus: PLC/IP3, DAG/PK-C pathway triggered by CaM

In Oncopeltus fasciatus, evidence shown here indicates it is calmodulin (CaM) that activates phospholipase-C (PLC), beginning a signalling pathway necessary for endocytic uptake of yolk precursor molecules. Epithelial cell-produced CaM, transported to oocytes via gap junctions, has been shown to be required for receptor-mediated endocytic uptake of vitellogenins (Vgs, the protein precursors of yolk). To determine if CaM was directly or indirectly stimulating the phospholipase-C (PLC) signalling cascade and thus controlling Vg endocytosis we used a series of molecules known to inactivate various elements of the pathway. W-7 prevents CaM from interacting with other molecules. Neomycin isolates PIP₂ from PLC. U-73122 directly inactivates PLC. 2-APB blocks IP₃ receptors which would otherwise cause release of Ca²⁺. Verapamil and CdCl₂ block Ca²⁺ release channels. Staurosporin and calphostin are inhibitors of PK-C. 1-Hexadecyl-2-acetyl glycerol (HAG) binds to diacylglycerol (DAG). Through the use of these antagonists we show here that: (1) the activation of phospholipase-C in this system requires CaM. (2) Stimulated phospholipase-C converts PIP₂ into IP₃ and DAG. (3) IP₃ causes increase in cytosolic Ca²⁺. (4) DAG and Ca²⁺ each stimulate phosphokinase-C, resulting in endocytosis of Vgs.     

Stimulation of phosphoinositide degradation and phosphatidylinositol-4-phosphate phosphorylation by GTP exclusively in plasma membrane of rat brain

The effect of GTP on the hydrolysis of [³H]phosphatidyinositol (PI), [³H]phosphatidylinositol-4-phosphate (PIP) and [³H]phosphatidylinositol-4,5-bisphosphate (PIP₂) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP₂ was actively degradted, opposite to PI and PIP which require Ca²⁺ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP₂ exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca²⁺ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP₂. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP₃ through the activation of phospholipase C acting on PIP₂ exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP₂ due to stimulation of PIP kinase.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.     

Intracellular mobilization of Ca by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca²⁺ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca²⁺ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist, and an L- or T-type Ca²⁺ channel blocker. The T-type Ca²⁺ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca²⁺-free medium, indicating that the source of Ca²⁺ is an intracellular reservoir. The IP₃R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca²⁺ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca²⁺ by activating IP₃R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP₃.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

U-73122 inhibits carbachol-induced increases in [Ca2+]i,IP3, and insulin release in β-TC3 cells

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca²⁺]_i, and IP₃ in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca²⁺]_i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP₃) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca²⁺]_i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca²⁺]_i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP₃, [Ca²⁺]_i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.     

Cyanide-stimulated inositol 1,4,5-trisphosphate formation: An intracellular neurotoxic signaling cascade

Cyanide-induced neurotoxicity is associated with altered cellular Ca²⁺ homeostasis resulting in sustained elevation of cytosolic Ca²⁺. In order to characterize the effect of cyanide on intracellular signaling mechanisms, the interaction of KCN with the inositol 1,4,5-triphosphate Ca²⁺ signaling system was determined in the PC12 cell line. KCN in the concentration range of 1.0–100 μM produced a rapid rise in intracellular IP₃ levels (peak level occurred within 60 sec); 10 μM KCN elevated intracellular levels of IP₃ to 148% of control levels. This response was mediated by phospholipase C (PLC) since U73122, a specific PLC inhibitor, blocked the response. Removal of Ca²⁺ from the incubation medium and chelation of intracellular Ca²⁺ with BAPTA partially attenuate the cyanide-stimulated IP₃ generation, showing that the response is partially Ca²⁺ dependent. Also, treatment of cells with nifedipine or LaCl₃, Ca²⁺ channel blockers, partially blocked the generation of IP₃. This study shows that cyanide in concentrations as low as 1 μM stimulates IP₃ generation that may be mediated by receptor and nonreceptor IP₃ production since they have differential dependence on Ca²⁺. It is proposed that this response is an early intracellular signaling action that can contribute to altered Ca²⁺ homeostasis characteristic of cyanide neurotoxicity. © 1997 John Wiley & Sons, Inc.     

Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein,Giα

In a previous report we shwed that glucocorticoed inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Giα levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Giα. In order to establish the nature of the coupliing between cytosolic Giα and cytosolic PLC we examined the effects of Protein activators, and inhibitors on cytosolic PLC activity from rat spenocytes and the rat lymphoma cell line Nb 2, with [³H] PI and [³H]PIP₂ as substrates. (1) Neither GTP nor its nonhydrolyzable analogue, GTPγS, at 100 μm had any effect on the calcium stimulated as well as the basal PLC activity. (2) Howevr, affinity purified antibodies to Giα1 and Giα2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP₂ as substrate, soluble PLC activity was inhibited 50–70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. (3)Administration of Giα1 antisense oligonucleotides to splenocytes for 48 h produced 25–40% decrease in cytosolic Giα1 levels compared to control. The soluble PLC activity with both PI and PIP₂ as substrates was also reduced by 25–50% compared to control conditions. This suggest that cytosolic Giα is associated with the activation of splenocyte soluble PLC. (4) Pertussis toxin administered in vivo sugnificantly reduced cytosolic Giα immunoreactivity and soluble PLC activiry when PI was used as substrate, providing additional evidence that cytosolic Giα is associated with the activation of splencyte soluble PLC. (5) Another agent that has beeen used extensively to define G-protein coupled processes is NaF/AlCl₃. NaF(4mM; with or without AlCl₃ inhibited soluble PLC activity with PIP₂ as substrate, in contrast ot the stimulatory effect that has been reported in the activation of membrane PLC. 6) because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperzine (50 μm, TFP), an inhibitor of protein phosphatase 2B; TFP (50 μm) signigicantly inhibited soluble PLC activity PI was used as substrate. These results suggest a direct involvement of cytosolic Giα in the activation of soluble PLC form splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Giα coupled PLC is addressed in the second paper.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP₃ accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP₃ accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP₃ in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.     

Departure gate of acidic Ca2+ confirmed

More potent, but less known than IP₃ that liberates Ca²⁺ from the ER, NAADP releases Ca²⁺ from acidic stores. The notion that TPC channels mediate this Ca²⁺ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P₂‐activated Na⁺ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P₂. They attribute the failure of others to observe TPC‐dependent NAADP‐induced Ca²⁺ release in vivo to inadequate mouse models that retain partial TPC function.     

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca²⁺-protein kinase C (Ca²⁺-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP₂), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca²⁺-PKC and the PIP₂-binding peptide of MARCKS modulate the level of free PIP₂, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP₂ and thereby inhibits PI3K binding to the membrane. In the on state, Ca²⁺-PKC phosphorylation of the MARCKS peptide reverses the PIP₂ sequestration, thereby releasing multiple PIP₂ molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca²⁺-PKC-stimulated release of free PIP₂ may well regulate the membrane association of other PIP₂-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.     

The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl₃ (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca²⁺]_i was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl₃ could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca²⁺]_i could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP₃ receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq–PLC–IP₃ pathway.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

The involvement of the cytoskeleton in regulating IP3 receptor-mediated internal Ca2+ release in human blood platelets.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP₃ in regulating internal Ca²⁺ release, and to measure IP₃ binding to its receptor. Our results indicate that platelet vesicles release Ca²⁺ as early as 3 seconds after IP₃ addition. Using [³H]IP₃, we have found that platelets contain a single class of high affinity IP₃ binding sites with a Kd of ～0.20 (± 0.01) nM. Immuno-blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP₃ receptor. Immunofluorescence staining data indicate that the IP₃ receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP₃ binding and IP₃-induced Ca²⁺ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP₃ binding and IP₃ receptor-mediated Ca²⁺ release during platelet activation.     

Conjugated linoleic acid-induced apoptosis in mouse mammary tumor cells is mediated by both G protein coupled receptor-dependent activation of the AMP-activated protein kinase pathway and by oxidative stress

Conjugated linoleic acid (CLA) has shown chemopreventive activity in several tumorigenesis models, in part through induction of apoptosis. We previously demonstrated that the t10,c12 isomer of CLA induced apoptosis of TM4t mouse mammary tumor cells through both mitochondrial and endoplasmic reticulum (ER) stress pathways, and that the AMP-activated protein kinase (AMPK) played a critical role in the apoptotic effect. In the current study, we focused on the upstream pathways by which AMPK was activated, and additionally evaluated the contributing role of oxidative stress to apoptosis. CLA-induced activation of AMPK and/or induction of apoptosis were inhibited by infection of TM4t cells with an adenovirus expressing a peptide which blocks the interaction between the G protein coupled receptor (GPCR) and Gα_q, by the phospholipase C (PLC) inhibitor U73122, by the inositol trisphosphate (IP₃) receptor inhibitor 2-APB, by the calcium/calmodulin-dependent protein kinase kinase α (CaMKK) inhibitor STO-609 and by the intracellular Ca²⁺ chelator BAPTA-AM. This suggests that t10,c12-CLA may exert its apoptotic effect by stimulating GPCR through Gα_q signaling, activation of phosphatidylinositol-PLC, followed by binding of the PLC-generated IP₃ to its receptor on the ER, triggering Ca²⁺ release from the ER and finally stimulating the CaMKK–AMPK pathway. t10,c12-CLA also increased oxidative stress and lipid peroxidation, and antioxidants blocked its apoptotic effect, as well as the CLA-induced activation of p38 MAPK, a downstream effector of AMPK. Together these data elucidate two major pathways by which t10,c12-CLA induces apoptosis, and suggest a point of intersection of the two pathways both upstream and downstream of AMPK.     

Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C

We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca²⁺ concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP₂), with a higher affinity for PIP₂. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein''s Ca²⁺ sensitivity and substrate preference, demonstrating the role of this motif in Ca²⁺ regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP₂. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP₂ and a contribution to the inositol 1,4,5-trisphosphate (IP₃)/diacylglycerol (DAG) pathway.     

UV light activates a Gαq/11-coupled phototransduction pathway in human melanocytes

While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C β (PLCβ) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca²⁺, and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gα_q/11 dependent. Activation of Gα_q/11/PLCβ signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP₂) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP₃). We found that PIP₂ regulated TRPA1-mediated photocurrents, and IP₃ stimulated intracellular Ca²⁺ release. The UVR-elicited Ca²⁺ response appears to involve both IP₃-mediated release from intracellular stores and Ca²⁺ influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gα_q/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye.