A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P₂ and PI(3,4,5)P₃. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTEN_CiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTEN_CiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P₃, but not toward PI(4,5)P₂. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P₂ and PI(3,4,5)P₃ of PTEN_CiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P₃ in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.     

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P₂) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P₂-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P₂ is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P₂ cross links two parts of the channel, enabling their coordinated movement during channel gating.     

The PDZ2 domain of zonula occludens-1 and -2 is a phosphoinositide binding domain

Zonula occludens proteins (ZO) are postsynaptic density protein-95 discs large-zonula occludens (PDZ) domain-containing proteins that play a fundamental role in the assembly of tight junctions and establishment of cell polarity. Here, we show that the second PDZ domain of ZO-1 and ZO-2 binds phosphoinositides (PtdInsP) and we identified critical residues involved in the interaction. Furthermore, peptide and PtdInsP binding of ZO PDZ2 domains are mutually exclusive. Although lipid binding does not seem to be required for plasma membrane localisation of ZO-1, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ₂) binding to the PDZ2 domain of ZO-2 regulates ZO-2 recruitment to nuclear speckles. Knockdown of ZO-2 expression disrupts speckle morphology, indicating that ZO-2 might play an active role in formation and stabilisation of these subnuclear structures. This study shows for the first time that ZO isoforms bind PtdInsPs and offers an alternative regulatory mechanism for the formation and stabilisation of protein complexes in the nucleus.     

The phosphoinositide PI(3,5)P2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P₂. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P₂ did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P₂ elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P₂ was applied to hTPC2 expressed in baker’s yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P₂-mediated activation and Na⁺ selectivity of mammalian TPC2.     

Signalling roles of mammalian phospholipase D1 and D2

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, protein kinase Cα as well as the lipid, phosphatidylinositol [4, 5] bisphosphate (PIP₂). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase, an enzyme that synthesises PIP₂. A signalling role of phospholipase D via PA and indirectly via PIP₂ in regulating membrane traffic and actin dynamics is indicated by the available data. Received 25 April 2001; received after revision 15 June 2001; accepted 15 June 2001     

P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells

We investigated the expression of P2X₄ and P2X₆ receptors on human umbilical vein endothelial cells (HUVECs) and found that both P2X receptor subtypes on plasma membranes are largely restricted to areas of cell-cell contact. Co-labelling experiments at the confocal and electron microscopy levels revealed that P2X₄ and P2X₆ receptors are strongly co-localised with the cell adhesion molecule VE-cadherin. The P2X₄ and P2X₆ receptors on plasma membranes at cellular junctions are rapidly (within 5 min) internalised specifically after decreasing extracellular [Ca²⁺]. Disruption of microfilaments, microtubules and integrin-mediated adhesion or stimulation of P2 receptors with ATP did not alter P2X₄ and P2X₆ receptor expression on HUVEC plasma membranes. Membraneous P2X₄ and P2X₆ receptors resisted extraction with Triton-X 100, whereas cytoplasmic P2X receptors were Triton-X 100 soluble. P2X₄ receptors, but not P2X₆ receptors, could be co-immunoprecipitated with VE-cadherin and vice versa. We conclude that P2X₄ and P2X₆ receptors are associated with VE-cadherin at HUVEC adherens junctions. Received 15 March 2002; revised 15 March 2002; accepted 19 March 2002     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

Lactacystin-induced apoptosis of cultured mouse cortical neurons is associated with accumulation of PTEN in the detergent-resistant membrane fraction

The tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate [PtdIns(3,4,5)P₃]. Implicit in this notion is that PTEN needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P₃. However, the recruitment of PTEN to the plasma membrane is not fully understood. Here, we demonstrate PTEN accumulation in the detergent-insoluble fraction of neuronal cells in response to treatment by the proteasome inhibitor lactacystin. First, lactacystin induces apoptosis and the activation of caspase-3 in cultured cortical neurons. Second, PTEN undergoes proteolysis to form a truncated 50-kDa form that lacks parts of its C-terminal tail. Third, the truncated PTEN is stably associated with the detergent-insoluble fraction in which the plasma membrane marker protein flotillin-1 resides. Taken together, our results suggest that truncation and accumulation of PTEN to the detergent-insoluble membrane fraction are two events associated with the apoptotic signals of the proteasome inhibitor in cortical neurons.Received 24 March 2004; received after revision 26 May 2004; accepted 5 June 2004     

Flippases: still more questions than answers

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P₄-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The β-subunit of P₄-ATPases shows unexpected similarities between the β- and γ-subunits of the Na⁺/K⁺-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P₄-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P_5A-ATPases are functional homologues of P₄-ATPases. Received 19 June 2008; received after revision 31 July 2008; accepted 15 August 2008     

Biochemical events associated with rapid cellular damage during the oxygen-and calcium-paradoxes of the mammalian heart

Summary The O_2– and Ca²⁺-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H⁺ and e^– and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca²⁺ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca²⁺ or by raising [Ca²⁺]_i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca²⁺-paradox by the membrane perturbation associated with removal of extracellular Ca²⁺; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca²⁺]_i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca²⁺-paradox which continues under anoxia. Massive entry of Ca²⁺ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Proteomic analysis of low-abundant integral plasma membrane proteins based on gels

To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na₂CO₃, chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fiftyseven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin. Received 15 March 2006; received after revision 17 May 2006; accepted 10 June 2006 L.-J. Zhang and X.-e Wang are contributed equally to this work.     

A quick and modified Winkler-method for measuring O2-concumption of aquatic animals

Summary A modified procedure for measuring O₂-consumption, based on Winkler-method, is described. Instead of KI and HCl (or H₂SO₄) triphenylmethane-dye leukoberbelinblue I and citric acid are used.Acknowledgment. Md. B. expresses his gratitude to DAAD (German Academic Exchange Service, Bonn) for awarding him a post-doctoral fellowship, during the tenure of which this investigation was carried out.     

Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence

Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y₂ (rP2Y₂) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca²⁺]_i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y₂-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin- mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.Received 8 February 2005; received after revision 18 March 2005; accepted 11 April 2005     

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE₂ increased [³H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE₂ increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE₂ obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE₂-induced cell cycle regulatory protein expression and thymidine incorporation. PGE₂ caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE₂-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells. Received 30 January 2009; received after revision 03 March 2009; accepted 10 March 2009     

Transduction of the chemotactic cAMP signal across the plasma membrane ofDictyostelium cells

AggregatingDictyostelium cells secrete cAMP during cell aggregation. cAMP induces two fast responses, the production of more cAMP (relay) and directed cell locomotion (chemotaxis). Extracellular cAMP binds to G-protein-coupled receptors leading to the activation of second messenger pathways, including the activation of adenylyl cyclase, guanylyl cyclase, phospholipase C and the opening of plasma membrane Ca²⁺ channels. Many genes encoding these sensory transduction proteins have been cloned and null mutants of nearly all components have been characterized in detail. Undoubtedly, activation of adenylyl cyclase is the most complex, involving G-proteins, a soluble protein called CRAC and components of the MAP kinase pathway. Null mutants in this pathway do not aggregate, but can exhibit chemotaxis and develop normally when supplied with exogenous cAMP. The pathways leading to the activation of phospholipase C were identified, but unexpectedly, deletion of the phospholipase C gene has no effect on chemotaxis and development, nor on intracellular Ins(1,4,5)P₃ levels; the metabolism of this second messenger will be discussed in some detail. Activation of guanylyl cyclase is G-protein-dependent and essential for chemotaxis. Analysis of a collection of chemotactic mutants reveals that most mutants are defective in either the production or intracellular detection of cGMP, thereby placing this second messenger at the center of chemotactic signal transduction. Analysis of the cAMP-mediated opening of plasma membrane calcium channels in signal transduction mutants suggests that it has two components, one that depends on G-proteins and intracellular cGMP and one that is G-protein-independent.     

Substance P-induced fibrinolysis in the forearm of healthy humans

Physiological saline with or without substance P (50 ng/ml) was infused into the humeral artery in 6 healthy males. Indices of fibrinolytic activity (whole blood diluted lysis time, euglobulin lysis time, lysis areas in non-heated fibrin plates produced by plasma or euglobulin precipitate, plasminogen plasma levels, ₂-macroglobulin, Cl-inhibitor, and ₂-antiplasmin) were evaluated in the homolateral antecubital vein before and after 5 min of substance P or saline infusion. After substance P the fibrinolytic activity increased, as can be seen from the shortening of lysis times (p<0.01) and enlargement of the lysis areas (p<0.01). A reduction of plasminogen plasma levels (p<0.01), associated with a decrease in ₂-anitplasmin (p<0.01), was also found. Alpha₂-macroglobulin and Cl-inhibitor were instead unaltered by the peptide. The saline infusion, on the other hand, was unable to modify any of the examined indices. We concluded that exogenous substance P given intra-arterially increases fibrinolytic activity in locally-sampled venous blood through a mechanism which remains to be elucidated.     

Transthyretin binds to glucose-regulated proteins and is subjected to endocytosis by the pancreatic β-cell

Transthyretin (TTR) is a functional protein in the pancreatic β-cell. It promotes insulin release and protects against β-cell death. We now demonstrate by ligand blotting, adsorption to specific magnetic beads, and surface plasmon resonance that TTR binds to glucose-regulated proteins (Grps)78, 94, and 170, which are members of the endoplasmic reticulum chaperone family, but Grps78 and 94 have also been found at the plasma membrane. The effect of TTR on changes in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) was abolished if the cells were treated with either dynasore, a specific inhibitor of dynamin GTPase that blocks clathrin-mediated endocytosis, or an antibody against Grp78, that prevents TTR from binding to Grp78. The conclusion is that TTR binds to Grp78 at the plasma membrane, is internalized into the β-cell via a clathrin-dependent pathway, and that this internalization is necessary for the effects of TTR on β-cell function.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.