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1. We have compared the effects of diflunisal on prostaglandin (PG) synthetase from human platelets and sheep seminal vesicles (SSV) by measuring malonaldehyde, PG products, and rabbit aorta contraction. 2. Aspirin inhibits PG synthetase by covalently acetylating the enzyme. Inhibition is dependent on time and aspirin concentration. Aspirin is 37-fold more potent in inhibiting enzyme in human platelets compared with enzyme from SSV. 3. Diflunisal inhibits PG synthetase from intact human platelets and SSV equally (50% inhibition at 3-5 micronM). The drug does not covalently modify the enzyme, and inhibition is not time dependent. Diflunisal probably acts at a site similar to aspirin since the drug will inhibit acetylation of the enzyme by aspirin. 4. The results suggest that diflunisal is relatively less inhibitory to platelet function than is aspirin. [ABSTRACT FROM AUTHOR]

The title of this article is also its punch line. The thesis that I will prove is that every adult, with a few exceptions, should take one 325 mg aspirin tablet each day. The drug is extraordinary and is beneficial in myriad ways. In this dosage the toxicity of the treatment is minimal. Since the drug is sold "over the counter", not requiring prescription, it is cheap and its benefits are easily underestimated. I do not use extensive reference citations; but just tell the story of aspirin., (Copyright © 2013. Published by Elsevier Ltd.)

The myotubularins are a large family of inositol polyphosphate 3-phosphatases that, despite having common substrates, subsume unique functions in cells that are disparate. The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] at the D-3 position. Seven members are inactive because they lack the conserved cysteine residue in the CX(5)R motif required for activity. We studied a subfamily of homologous myotubularins, including myotubularin-related protein 6 (MTMR6), MTMR7, and MTMR8, all of which dimerize with the catalytically inactive MTMR9. Complex formation between the active myotubularins and MTMR9 increases their catalytic activity and alters their substrate specificity, wherein the MTMR6/R9 complex prefers PtdIns(3,5)P(2) as substrate; the MTMR8/R9 complex prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of MTMR6 toward PtdIns(3,5)P(2) by over 30-fold, and enhanced the activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 increased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns(3,5)P(2) and PtdIns(3)P, respectively. In cells, the MTMR6/R9 complex significantly increases the cellular levels of PtdIns(5)P, the product of PI(3,5)P(2) dephosphorylation, whereas the MTMR8/R9 complex reduces cellular PtdIns(3)P levels. Consequentially, the MTMR6/R9 complex serves to inhibit stress-induced apoptosis and the MTMR8/R9 complex inhibits autophagy.

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells.

Osteoporosis is a multifactorial genetic disease characterized by reduction of bone mass due to dysregulation of osteoclast differentiation or maturation. Herein, we identified a regulator of osteoclastogenesis, the murine homolog of inositol polyphosphate 4-phosphatase type IIα (Inpp4bα). Expression of Inpp4bα is detected from early osteoclast differentiation to activation stage. Targeted expression of native Inpp4bα ex vivo repressed whereas phosphatase-inactive Inpp4bα stimulated osteoclast differentiation. Inpp4bα acts on intracellular calcium level that modulates NFATc1 nuclear translocation and activation. In vivo mice deficient in Inpp4b displayed increased osteoclast differentiation rate and potential resulting in decreased bone mass and osteoporosis. Importantly, INPP4B in human was identified as a susceptibility locus for osteoporosis. This study defined Inpp4b as a major modulator of the osteoclast differentiation and as a gene linked to variability of bone mineral density in mice and humans., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas.

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

Phosphotidylinositol (PtdIns) signaling is tightly regulated both spatially and temporally by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a key regulatory enzyme at the branch point for the synthesis of inositol hexakisphosphate (IP(6)), an intracellular signaling molecule implicated in the regulation of ion channels, endocytosis, exocytosis, transcription, DNA repair, and RNA export from the nucleus. IP(6) also has been shown to be an integral structural component of several proteins. We have generated a mouse strain harboring a beta-galactosidase (betagal) gene trap cassette in the second intron of the Itpk1 gene. Animals homozygous for this gene trap are viable, fertile, and produce less ITPK1 protein than wild-type and heterozygous animals. Thus, the gene trap represents a hypomorphic rather than a null allele. Using a combination of immunohistochemistry, in situ hybridization, and betagal staining of mice heterozygous for the hypomorphic allele, we found high expression of Itpk1 in the developing central and peripheral nervous systems and in the paraxial mesoderm. Examination of embryos resulting from homozygous matings uncovered neural tube defects (NTDs) in some animals and axial skeletal defects or growth retardation in others. On a C57BL/6 x 129(P2)Ola background, 12% of mid-gestation embryos had spina bifida and/or exencephaly, whereas wild-type animals of the same genetic background had no NTDs. We conclude that ITPK1 is required for proper development of the neural tube and axial mesoderm.

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P(2) is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 ((-/-)MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to (+/+)MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser(473) and Thr(308)-Akt phosphorylation and activation of Akt-dependent signalling in (-/-)MEFs, relative to (+/+)MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed (-/-)MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.

The field of inositol signaling has expanded greatly in recent years. Given the many reviews on phosphoinositide kinases, we have chosen to restrict our discussion to inositol lipid hydrolysis focused on the phosphatases and a brief mention of the lipase isoforms. We also discuss recent discoveries that link mutations in phosphoinositide phosphatases to disease.

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.

Inositol pyrophosphates were, until recently, without clearly defined functions. Two recent papers in Science have now clearly defined a function for an IP(7) pyrophosphate (inositol hexaphosphate with one pyrophosphate) that is the product of the enzyme encoded by the Vip1 gene in Saccharomyces cerevisiae. This IP(7) with a pyrophosphate tentatively assigned to be on either the 4 or 6 position is a cofactor that is required for inactivating the cyclin-cyclin-dependent kinase complex of Pho80, Pho81, and Pho85. Inhibition of the kinase results in the nuclear translocation of Pho4, which is a transcription factor that promotes expression of genes required for phosphate assimilation under conditions of low phosphate. When grown in low-phosphate media, IP(7) accumulates, which leads to the expression of genes involved in the acquisition of phosphate.

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P(2)), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNA-damaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.

The human inositol phosphate multikinase (IPMK, 5-kinase) has a preferred 5-kinase activity over 3-kinase and 6-kinase activities and a substrate preference for inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) over inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). We now report that the recombinant human protein can catalyze the conversion of inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P4) to Ins(1,3,4,5,6)P5 in vitro; the reaction product was identified by HPLC to be Ins(1,3,4,5,6)P5. The apparent Vmax was 42 nmol of Ins(1,3,4,5,6)P5 formed/min/mg protein, and the apparent Km was 222 nM using Ins(1,3,4,6)P4 as a substrate; the catalytic efficiency was similar to that for Ins(1,4,5)P3. Stable over-expression of the human protein in HEK-293 cells abrogates the in vivo elevation of Ins(1,4,5,6)P4 from the Salmonella dublin SopB protein. Hence, the human 5-kinase may also regulate the level of Ins(1,4,5,6)P4 and have an effect on chloride channel regulation.

Numerous inositol polyphosphate 5-phosphatases catalyze the degradation of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P(2)) to phosphatidylinositol-4-phosphate (PtdIns-4-P). An alternative pathway to degrade PtdIns-4,5-P(2) is the hydrolysis of PtdIns-4,5-P(2) by a 4-phosphatase, leading to the production of PtdIns-5-P. Whereas the bacterial IpgD enzyme is known to catalyze this reaction, no such mammalian enzyme has been found. We have identified and characterized two previously undescribed human enzymes, PtdIns-4,5-P(2) 4-phosphatase type I and type II, which catalyze the hydrolysis of PtdIns-4,5-P(2) to phosphatidylinositol-5-phosphate (PtdIns-5-P). Both enzymes are ubiquitously expressed and localize to late endosomal/lysosomal membranes in epithelial cells. Overexpression of either enzyme in HeLa cells increases EGF-receptor degradation upon EGF stimulation.

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor alpha (TNF(alpha))-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFalpha-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFalpha-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFalpha-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis.

Many functions have been suggested for inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), including mRNA export, nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsP6 is necessary for in vivo survival. We previously isolated a cDNA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsP5) 2-kinase (2-kinase), the enzyme that converts InsP5 to InsP6. We used the sequence to search the BayGenomics databases and identify an ES cell line (XA232) that has a gene trap construct embedded in the 2-kinase gene. We obtained a mouse from this line, produced heterozygotes, and confirmed that the heterozygotes contain the trapping construct and have diminished 2-kinase activity. Breeding the XA232 heterozygotes produced no homozygous offspring; thus, loss of 2-kinase is lethal in mice. Dissections of embryonic day-8.5 uteri yielded no homozygous embryos; thus, the mice die before day 8.5 postcoitum. The gene trap construct contains a beta-galactosidase/neomycin reporter gene, allowing us to stain heterozygotes for beta-galactosidase to determine tissue-specific expression of 2-kinase protein. 2-kinase is expressed in the hippocampus, the cortex, the Purkinje layer of the cerebellum in the brain, in cardiomyocytes, and in the testes of adult mice. At day 9.5 postcoitum, 2-kinase was expressed in the notochord, the ventricular layer of the neural tube, and the myotome of the somites. Intense staining was also seen in the yolk sac, suggesting that InsP6 is necessary for yolk sac development or function. Furthermore, failure of yolk sac development or function is consistent with the early lethality of 2-kinase embryos.

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Ialpha inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P2, forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P2, and C2-domain-deletion mutants lost PtdIns(3,4)P2 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations.

Inositol hexakisphosphate and other inositol high polyphosphates have diverse and critical roles in eukaryotic regulatory pathways. Inositol 1,3,4-trisphosphate 5/6-kinase catalyzes the rate-limiting step in inositol high polyphosphate synthesis in animals. This multifunctional enzyme also has inositol 3,4,5,6-tetrakisphosphate 1-kinase and other activities. The structure of an archetypal family member, from Entamoeba histolytica, has been determined to 1.2 A resolution in binary and ternary complexes with nucleotide, substrate, and product. The structure reveals an ATP-grasp fold. The inositol ring faces ATP edge-on such that the 5- and 6-hydroxyl groups are nearly equidistant from the ATP gamma-phosphate in catalytically productive phosphoacceptor positions and explains the unusual dual site specificity of this kinase. Inositol tris- and tetrakisphosphates interact via three phosphate binding subsites and one solvent-exposed site that could in principle be occupied by 18 different substrates, explaining the mechanisms for the multiple specificities and catalytic activities of this enzyme.

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

With this issue of the JCI, we celebrate the 80th anniversary of the Journal. While 80 years is not a century, we still feel it is important to honor what the JCI has meant to the biomedical research community for 8 decades. To illustrate why the JCI is the leading general-interest translational research journal edited by and for biomedical researchers, we have asked former JCI editors-in-chief to reflect on some of the major scientific advances reported in the pages of the Journal during their tenures.

The subcellular localization of Ocrl, the inositol polyphosphate 5-phosphatase that is mutated in Lowe syndrome, was investigated by fluorescence microscopy. Ocrl was localized to endosomes and Golgi membranes along with clathrin, giantin, the mannose 6-phosphate receptor, transferrin, and the early endosomal antigen 1 endosomal marker in fixed cells. The endosomal localization of Ocrl was confirmed by live-cell time-lapse microscopy in which we monitored the dynamics of Ocrl on endosomes. GST binding assays show that Ocrl interacts with the clathrin terminal domain and the clathrin adaptor protein AP-2. Our findings suggest a role for Ocrl in endosomal receptor trafficking and sorting.

The protein Gle1 is required for export of mRNAs from the nucleus to the cytoplasm in both lower and higher eukaryotic cells. In human (h) cells, shuttling of hGle1 between the nucleus and cytoplasm is essential for bulk mRNA export. To date, no hGle1-interacting proteins have been reported and the mechanism by which hGle1 interacts with the nuclear pore complex (NPC) and mediates export is unknown. To identify proteins that can interact with hGle1, a genome-wide yeast two-hybrid screen was performed. Three potential hGle1-interacting partners were isolated, including clones encoding the C-terminal region of the NPC protein hNup155. This interaction between hGle1 and full-length hNup155 was confirmed in vitro, and deletion analysis identified the N-terminal 29 residues of hGle1 as the hNup155-binding domain. Experiments in HeLa cells confirmed that the nuclear rim localization of the major hGle1 protein variant (hGle1B) was dependent on the presence of these 29 N-terminal residues. This suggests that this domain of hGle1 is necessary for targeting to the NPC. This work also characterizes the first domain in hNup155, a 177 C-terminal amino acid span that binds to hGle1. The mutual interaction between hGle1 and the symmetrically distributed nuclear pore protein Nup155 suggests a model in which hGle1's association with hNup155 may represent a step in the Gle1-mediated mRNA export pathway.

Tumor necrosis factor receptor 1 (TNF-R1) signaling elicits a wide range of biological responses, including inflammation, proliferation, differentiation, and apoptosis. TNF-R1 activates both caspase-mediated apoptosis and NF-kappaB transcription of anti-apoptotic factors. We now report a link between the TNF-R1 and inositol phosphate signaling pathways. We observed that overexpression of inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) inhibited apoptosis induced by TNFalpha. The anti-apoptotic effect by 5/6-kinase is not attributable to NF-kappaB activation, as no changes were detected in the levels of NF-kappaB DNA binding, IkappaBalpha degradation, or anti-apoptotic factors, such as x-linked inhibitor of apoptosis protein. Decreased expression of 5/6-kinase by RNA interference rendered HeLa cells more susceptible to TNFalpha-induced apoptosis. Overexpression of 5/6-kinase in human embryonic kidney 293 cells inhibited TNFalpha-induced activation of caspases-8, -3, and -9, BID, and poly(ADP-ribose) polymerase. However, 5/6-kinase did not protect against Fas-, etoposide-, or cycloheximide-induced apoptosis. Further, 5/6-kinase protected against apoptosis induced by the overexpression of TNF-R1-associated death domain but not Fas-associated death domain. Therefore, we suggest that 5/6-kinase modifies TNFalpha-induced apoptosis by interfering with the activation of TNF-R1-associated death domain.

Myotubularin-related protein 7 (MTMR7) is a member of the myotubularin (MTM) family. The cDNA encoding the mouse MTMR7 contains 1,983 bp, and the predicted protein has a deduced molecular mass of 75.6 kDa. Northern and Western blot analyses showed that MTMR7 is expressed mainly in brain and mouse neuroblastoma N1E-115 cells. Recombinant MTMR7 dephosphorylated the D-3 position of phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate [Ins(1,3)P2]. The substrate specificity of MTMR7 is different than other MTM proteins in that this enzyme prefers the water-soluble substrate. Immunofluorescence showed that MTMR7 is localized in Golgi-like granules and cytosol, and subcellular fractionation showed both cytoplasmic and membrane localization of MTMR7 in N1E-115 cells. An MTMR7-binding protein was found in an anti-MTMR7 immunoprecipitate from N1E-115 cells and identified as MTM-related protein 9 (MTMR9) by tandem mass spectrometry. The coiled-coil domain of MTMR9 was sufficient for binding to MTMR7. The binding of MTMR9 increased the Ins(1,3)P2 phosphatase activity of MTMR7. Our results show that MTMR7 forms a complex with MTMR9 and dephosphorylates phosphatidylinositol 3-phosphate and Ins(1,3)P2 in neuronal cells.

Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in myotubularin result in the human disease X-linked myotubular myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human myotubularin were identified. Association between 3-PAP and myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with myotubularin led to attenuation of the plasma membrane phenotype, associated with myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an "adapter" for myotubularin, regulating myotubularin intracellular location and thereby altering the phenotype resulting from myotubularin overexpression.

The COP9 signalosome (CSN) is a complex of eight proteins first identified as a repressor of plant photomorphogenesis. A protein kinase activity associated with the COP9 signalosome has been reported but not identified; we present evidence for inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) as a protein kinase associated with the COP9 signalosome. We have shown that 5/6-kinase exists in a complex with the eight-component COP9 signalosome both when purified from bovine brain and when transfected into HEK 293 cells. 5/6-kinase phosphorylates the same substrates as those of the COP9 signalosome, including IkappaBalpha, p53, and c-Jun but fails to phosphorylate several other substrates, including c-Jun 1-79, which are not substrates for the COP9-associated kinase. Both the COP9 signalosome- associated kinase and 5/6-kinase are inhibited by curcumin. The association of 5/6-kinase with the COP9 signalosome is through an interaction with CSN1, which immunoprecipitates with 5/6-kinase. In addition, the inositol kinase activity of 5/6-kinase is inhibited when in a complex with CSN1. We propose that 5/6-kinase is the previously described COP9 signalosome-associated kinase.

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.

Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells.

Phosphorylation of inositol 1,3,4-trisphosphate by inositol 1,3,4-trisphosphate 5/6-kinase is the first committed step in the formation of higher phosphorylated forms of inositol. We have shown that the eight proteins called the COP9 signalosome complex copurify with calf brain 5/6-kinase. Because the complex has been shown to phosphorylate c-Jun in vitro, we tested both the complex and 5/6-kinase and found that both are able to phosphorylate c-Jun and ATF-2 on serine/threonine residues. These findings establish a link between two major signal transduction systems: the inositol phosphates and the stress response system.

The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP shares significant sequence similarity with the protein and lipid 3-phosphatase myotubularin, and with several other members of the myotubularin gene family including SET-binding factor 1. However, unlike myotubularin, 3-PAP does not contain a consensus HCX(5)R catalytic motif. The 3-PAP sequence contains several motifs that predict interaction with proteins containing Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, members of the 14-3-3 family, as well as proteins with SET domains. Northern blot analysis identified two transcripts (5.5 kb and 2.5 kb) with highest abundance in human liver, kidney, lung, and placenta. 3-PAP immunoprecipitates isolated from platelet cytosol hydrolyzed the D3-phosphate from PtdIns(3)P and PtdIns 3,4-bisphosphate [PtdIns(3,4)P(2)]. However, insect cell-expressed 3-PAP recombinant protein was catalytically inactive, confirming our prior prediction that this polypeptide represents an adapter subunit.

SopB is an inositol phosphate phosphatase that is a virulence factor in Salmonella species. We have overexpressed SopB cDNA in a tetracycline-dependent system in human embryonic 293 cells, and used this model system to directly analyze the role of SopB in altering inositol metabolite levels in vivo. Addition of tetracycline to these cells resulted in the rapid induction of SopB expression, which was coincident with perturbations in the cellular levels of multiple soluble inositol phosphates. All of the changes induced by SopB expression were reversed within 24 h on removal of tetracycline from media. Specifically, cellular inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) levels were depleted within 4 to 6 h after inducing SopB expression. A transient rise in cellular inositol 1,4,5,6-tetrakisphosphate was also observed and was accompanied by increased chloride channel activity. This indicates that SopB alone is sufficient for changes in chloride channel function in cells infected with Salmonella organisms. Depletion of inositol phosphates, including InsP(5) and InsP(6) metabolites, was coincident with the accumulation of polyadenylated RNA in the nucleus. This suggested that a defect in nuclear export had occurred. Moreover, the penetrance of the export defect required localization of SopB to the nucleus. These results provide evidence that inositol phosphate productions may be required for efficient mRNA export in mammalian cells.

Megakaryocytes lacking transcription factor GATA-1 fail to complete maturation in vivo and hyperproliferate. To define how GATA-1 regulates megakaryocyte cell growth we searched for mRNA transcripts expressed in primary wild-type, but not GATA-1(-), megakaryocytes. One differentially expressed transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and also has lesser activity against soluble analogues of this lipid, inositol 3, 4-bisphosphate and inositol 1,3,4-triphosphate. Reintroduction of 4-Ptase I into both primary GATA-1(-) and wild-type megakaryocytes significantly retards cell growth, suggesting that absence of 4-Ptase I may contribute to the hyperproliferative phenotype of GATA-1(-) megakaryocytes. Overexpression of 4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken together, these data indicate that 4-Ptase I is a regulator of cell proliferation.