Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation.

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.

Myotubularin-related protein (MTMR) 9 determines the enzymatic activity, substrate specificity, and role in autophagy of MTMR8.

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.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers.

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.

Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase.

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.

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4-phosphatase-1.

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.

INO-4995 therapeutic efficacy is enhanced with repeat dosing in cystic fibrosis knockout mice and human epithelia.

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.

A discrete signaling function for an inositol pyrophosphate.

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.

Blasts from the past.

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.

An aspirin a day.

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.

Inositol polyphosphate 4-phosphatase B as a regulator of bone mass in mice and humans.

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.

Phosphoinositide phosphatases and disease.

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.

MTMR9 increases MTMR6 enzyme activity, stability, and role in apoptosis.

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.

Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.

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.

Type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced apoptosis.

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.

Inositol polyphosphate multikinase regulates inositol 1,4,5,6-tetrakisphosphate.

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.

Increased levels of inositol hexakisphosphate (InsP6) protect HEK293 cells from tumor necrosis factor (alpha)- and Fas-induced apoptosis.

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.

Disruption of the mouse inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expression.

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.