Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

PtdIns(4,5)P2 is not required for secretory granule docking

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co‐clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high‐resolution total internal reflection imaging of EGFP‐labeled PtdIns markers or syntaxin‐1 at secretory granule release sites in live insulin‐secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin‐1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin‐1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) by recruitment of a 5′‐phosphatase strongly inhibited Ca²⁺‐dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin‐1. Cell permeabilization by α‐toxin or formaldehyde‐fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P₂ accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin‐1 at the release site.      

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.     

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.     

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

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LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling,lysosomal sorting,and degradation

Lysosomal degradation is essential for the termination of EGF‐stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi‐vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF‐induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT‐0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P₂ and recruiting SNX5. PtdIns(4,5)P₂ and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P₂ signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.     

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis

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Inhibition of Papaya latex papain by photosensitive inhibitors. 1-(4,5-dimethoxy-2-nitrophenyl)-2-nitroethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

1-(4,5-Dimethoxy-2-nitrophenyl)-2-nitroethene (1) was shown to be an irreversible inhibitor of papain (EC 3.4.22.2), causing a complete inhibition (120 min preincubation, pH 8.0), assuming that it attached to Cys-25 at the active site of the enzyme (while a short preincubation time caused activation). Only partial inhibition of papain was achieved, however, with 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2), a compound synthesized in this work, which is also an irreversible inhibitor of papain. Since both compounds 1 and 2, and in each case of the inhibited enzyme, were 2-nitrobenzyl derivatives, they and the modified enzyme were expected to be photosensitive. Indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in a full recovery of the enzyme activity following inactivation with compound 1 (similar to our previous finding with -galactosidase) and up to 67% recovery following inhibition with compound 2.     

Fluorescence enhancement of europium (III) perchlorate by 1,10‐phenanthroline on the 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone complex and luminescence mechanism

A novel ligand, 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone was synthesized using a new method and its two europium (Eu) (III) complexes were synthesized. The compounds were characterized by elemental analysis, coordination titration analysis, molar conductivity, infrared, thermo gravimetric analyzer‐differential scanning calorimetry (TGA‐DSC), ¹H NMR and UV spectra. The composition was suggested as EuL₅ · (ClO₄)₃ · 2H₂O and EuL₄ · phen(ClO₄)₃ · 2H₂O (L = C₁₀H₇COCH₂SOC₆H₅). The fluorescence spectra showed that the Eu(III) displayed strong characteristic metal‐centered fluorescence in the solid state. The ternary rare earth complex showed stronger fluorescence intensity than the binary rare earth complex in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 1.49 times as strong as that of the binary system. The phosphorescence spectra were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Convergent regulation of the lysosomal two‐pore channel‐2 by Mg2+, NAADP,PI(3,5)P2 and multiple protein kinases

Lysosomal Ca²⁺ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca²⁺ signaling was the discovery of the two‐pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P₂ activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg²⁺ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg²⁺ specifically inhibited TPC2 outward current, whereas lysosomal Mg²⁺ partially inhibited both outward and inward currents in a lysosomal lumen pH‐dependent manner. Under controlled Mg²⁺, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P₂. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP‐mediated Ca²⁺ release in intact cells is regulated by Mg²⁺, PI(3,5)P₂, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP‐mediated Ca²⁺ signaling and link this pathway to Mg²⁺ homeostasis and MAP kinases, pointing to roles for lysosomal Ca²⁺ in cell growth, inflammation and cancer.     

Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum

The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.     

Control of P2X(2) channel permeability by the cytosolic domain

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.