Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

Degradation of IkappaBalpha in activated RAW264.7 cells is blocked by the phosphatidylinositol 3-kinase inhibitor LY294002

The mechanism by which lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) induces production of proinflammatory cytokines in murine macrophages, and the role of phosphatidylinositol 3-kinase (PI3-kinase) have not been well investigated. Activation of nuclear factor κB (NF-κB) is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. In this study we demonstrate that 2- (4-morpholinyl)-8-phenylchromone (LY294002), which inhibits PI3-kinase, specifically inhibited degradation of IκBα in RAW264.7 cells stimulated with interferon-γ (IFN-γ) plus LPS or IFN-γ plus PMA. To elucidate the importance of this activity in RAW264.7 cells, we examined tumor necrosis factor-α (TNF-α) and interleukin IL)-6 production in the activated cells. Pretreatment of the cells with LY294002 resulted in the inhibition of TNF-α and IL-6 production in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. Furthermore, LY294002 inhibited the production of nitric oxide NO) in RAW264.7 cells stimulated with IFN-γ plus LPS or IFN-γ plus PMA. LY294002 also inhibited inducible NO synthase (iNOS) mRNA expression in the activated RAW264.7 cells. In conclusion, the present results suggest that PI3-kinase is involved in the signal transduction pathway responsible for LPS- or PMA-mediated TNF-α and IL-6 production, and that LY294002 inhibits NO generation through blocking the degradation of IκBα in activated RAW264.7 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Insulin-like and non-insulin-like selenium actions in 3T3-L1 adipocytes

In insulin-sensitive 3T3-L1 adipocytes, selenium stimulates glucose transport and antilipolysis and these actions of selenium, like insulin actions, are sensitive to wortmanin, an inhibitor of phosphatidylinositol-3-kinase (PI3K). Selenium stimulates PI3K activity that is sustained up to 24 h. Selenium after 5-10 min increases tyrosine phosphorylation of selective cellular proteins, but after 24 h overall tyrosine phosphorylation is increased. Tyrosine phosphorylation of insulin receptor substrate 1 is detected when enriched by immunoprecipitation with anti-PI3K antibody. Selenium, however, does not stimulate insulin receptor tyrosine kinase activity. Selenium also increases phosphorylation of other insulin signaling proteins, including Akt and extracellular signal regulated kinases. Selenium-stimulated glucose transport is accompanied by increases in glucose transporter-1 content in the plasma membrane. These data are consistent with similar selenium action in glucose transport in 3T3-L1 fibroblasts expressing mainly GLUT1. In chronic insulin-induced insulin resistant cells, selenium unlike insulin fully stimulates glucose transport. In summary, selenium stimulates glucose transport and antilipolysis in a PI3K-dependent manner, but independent of insulin receptor activation. Selenium exerts both insulin-like and non-insulin-like actions in cells.     

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.     

Lipid modulation of ion channels through specific binding sites

Ion channel conformational changes within the lipid membrane are a key requirement to control ion passage. Thus, it seems reasonable to assume that lipid composition should modulate ion channel function. There is increasing evidence that this implicates not just an indirect consequence of the lipid influence on the physical properties of the membrane, but also specific binding of selected lipids to certain protein domains. The result is that channel function and its consequences on excitability, contractility, intracellular signaling or any other process mediated by such channel proteins, could be subjected to modulation by membrane lipids. From this it follows that development, age, diet or diseases that alter lipid composition should also have an influence on those cellular properties. The wealth of data on the non-annular lipid binding sites in potassium channel from Streptomyces lividans (KcsA) makes this protein a good model to study the modulation of ion channel structure and function by lipids. The fact that this protein is able to assemble into clusters through the same non-annular sites, resulting in large changes in channel activity, makes these sites even more interesting as a potential target to develop lead compounds able to disrupt such interactions and hopefully, to modulate ion channel function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates a variety of lysophospholipids including bis(monoacylglycero)phosphate

When the lysoglycerophospholipid (GPL) acyltransferase At1g78690 from Arabidopsis thaliana is over-expressed in Escherichiacoli a headgroup acylated GPL, acyl phosphatidylglycerol (PG), accumulates despite that in vitro this enzyme catalyzes the transfer of an acyl chain from acyl-CoA to the sn-2 position of 1-acyl phosphatidylethanolamine (PE) or 1-acyl PG to form the sn-1, sn-2, di acyl PE and PG respectively; it does not acylate PG to form acyl PG. To begin to understand why the overexpression of a lyso GPL acyltransferase leads to the accumulation of a headgroup acylated GPL in E. coli we investigated the headgroup specificity of At1g78690. Using membranes prepared from E. coli overexpressing At1g78690, we assessed the ability of At1g78690 to catalyze the transfer of acyl chains from acyl-coenzyme A to a variety of lyso GPL acyl acceptors including lyso-phosphatidic acid (PA), -phosphatidylcholine (PC), -phosphatidylserine (PC), -phosphatidylinositol (PI) and three stereoisoforms of bis(monoacylglycero)phosphate (BMP). The predicted products were formed when lyso PI and lyso PC were used as the acyl acceptor but not with lyso PC or lyso PA. In addition, At1g78690 robustly acylates two BMP isoforms with sn-2 and/or sn-2′ hydroxyls in the R-stereoconfiguration, but not the BMP isoform with the sn-2 and sn-2′ hydroxyls in the S-stereoconfiguration. This strongly suggests that At1g78690 is stereoselective for hydroxyls with R-stereochemistry. In addition, this robust acylation of BMPs by At1g78690, which yields acyl PG like molecules, may explain the mechanism by which At1g78690 so strikingly alters the lipid composition of E. coli.     

The role of erythropoietin and its receptor in growth,survival and therapeutic response of human tumor cells: From clinic to bench — a critical review

Recombinant human erythropoietin (rhEPO) has been used clinically to alleviate cancer- and chemotherapy-related anemia. However, recent clinical trials have reported that rhEPO also may adversely impact disease progression and survival. The expression of functional EPO receptors (EPOR) has been demonstrated in many human cancer cells where, at least in vitro, rhEPO can stimulate cell growth and survival and may induce resistance to selected therapies.     

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P₃ levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P₃ and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.