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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.

ITPK1 is the rate limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP6 and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP5 or IP6 in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.

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.

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.

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.

Many functions have been suggested for inositol 1,2,3,4,5,6-hexakisphosphate (InsP 6 ), including mRNA export, nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsP 6 is necessary for in vivo survival. We previously isolated a cDNA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsP 5 ) 2-kinase (2-kinase), the enzyme that converts InsP 5 to InsP 6 . 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 β-galactosidase/neomycin reporter gene, allowing us to stain heterozygotes for β-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 InsP 6 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.

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.

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.

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.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP6) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP6 by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP5). 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 (InsP5 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP6 or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP5 to InsP6/min/mg of protein (V max). The Michaelis-Menten constant for InsP5 was 0.4 μm and for ATP was 21 μm. Saccharomyces cerevisiae lackingIPK1 do not produce InsP6 and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP5 2-kinase in a yeastipk1 null strain restored the synthesis of InsP6 and rescued the gle1–2 ipk1–4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP5 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP6 synthesis in mammalian cells will allow for further studies of the InsP6 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.

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20– to 25–amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4) P 2 ]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4) P 2 production.

The SH2-containing inositol phosphatase, SHIP, often appears as multiple bands in anti-SHIP immunoblots. To characterize these bands, antisera were generated against the N-terminal (anti-N), mid-region (anti-M), and C-terminal (anti-C) portions of SHIP. Immunoprecipitation and immunoblotting studies showed that 145-, 135-, 125-, and 110-kD bands were detected in lysates from the murine hematopoietic cell line, DA-ER, with either anti-N or anti-M antisera, whereas only the 145- and 135-kD bands were recognized by the anti-C antiserum. This finding suggested that the smaller proteins might be C-terminal truncations of the full-length SHIP. To confirm this and determine if these proteins arose through alternate splicing or posttranslational cleavage, a 5′-hemagglutin (HA)-tagged full-length SHIP cDNA was expressed in these cells. We observed, via Western analysis with anti-HA antibodies, the same 4 bands with either anti-N or anti-M and only the 145- and 135-kD bands with anti-C immunoprecipitation. After interleukin-3 stimulation of HA-SHIP–expressing DA-ER cells, only the 145-kD form coprecipitated with Shc, raising the possibility that different forms of SHIP may have distinct intracellular sites. This was confirmed by subcellular fractionation, which showed that only the 110-kD form is present in the cytoskeleton of DA-ER cells. This 110-kD form possesses the same PIP3 5-ptase activity as the 145-kD form and can be generated from the latter in vitro by digestion with calpain. It is therefore possible that the different forms of SHIP are generated in vivo by calpain-mediated C-terminal truncations and perform distinct functions within hematopoietic cells.© 1998 by The American Society of Hematology.

Inositol polyphosphate 4-phosphatase (4-phosphatase) is a Mg2+-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 3,4-bisphosphate. We have isolated cDNA encoding a 105,257-Da protein that is 37% identical to the previously cloned 4-phosphatase. Recombinant protein was expressed in Escherichia coli and shown to hydrolyze all three 4-phosphatase substrates with enzymatic properties similar to the original enzyme. We designate the original 4-phosphatase and the new isozyme as inositol polyphosphate 4-phosphatase types I and II, respectively. 4-Phosphatase II is highly conserved with the human and rat enzymes having 90% amino acid identity. A conserved motif between 4-phosphatase I and II is the sequence CKSAKDRT that contains the Cys-Xaa5-Arg active site consensus sequence identified for other Mg2+-independent phosphatases. Northern blot analysis indicated that 4-phosphatase II is widely expressed with the highest levels occurring in the skeletal muscle and heart. In addition, cDNA encoding alternatively spliced forms of human 4-phosphatase I (107, 309 Da) and rat 4-phosphatase II (106,497 Da) were also isolated that encode proteins with a putative transmembrane domain near their C termini. These alternatively spliced forms were expressed as recombinant proteins in E. coli and SF9 insect cells and found to possess no detectable enzymatic activity suggesting that additional factors and/or processing may be required for these alternatively spliced isozymes.

We have sequenced and recombinantly expressed as a fusion protein an expressed sequence tag clone (GB Z25963) from Arabidopsis thaliana that represents the plant homologue of human inositol 1,3,4 trisphosphate 5/6-kinase. The 1365 base pair clone has an open reading frame of 960 base pairs that predicts a protein product of 36.2 kDa, with a pI of 6.1. There is no polyadenylation signal or poly (A) tail, suggesting that additional 3' sequence remains to be identified. The amino acid sequence is 30% identical to the human protein. There are several short regions with particularly high degrees of identity between the human and Arabidopsis protein sequences, and these may be useful in identifying the active site of the enzyme. The expressed sequence tag was expressed as a fusion protein in Escherichia coli, with a carboxyl terminal deletion removing one region of high identity between the two proteins. The protein product of this construct was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. The Arabidopsis enzyme produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 1:3, in contrast with the human enzyme which gives a product ratio of 3:1.

An inositol polyphosphate-5-phosphatase (SIP-110) that binds the SH3 domains of the adaptor protein GRB2 was produced in Sf9 cells and characterized. SIP-110 binds to GRB2 in vitro with a stoichiometry of 1 mol of GRB2/0.7 mol of SIP-110. GRB2 binding does not affect enzyme activity implying that GRB2 serves mainly to localize SIP-110 within cells. SIP-110 hydrolyses inositol (Ins)(1,3,4,5)P4 to Ins(1,3,4)P3. The enzyme does not hydrolyze Ins(1,4,5)P3 that is a substrate for previously described 5-phosphatases nor does it hydrolyze phosphatidylinositol (PtdIns)(4,5)P2. SIP-110 also hydrolyzed PtdIns(3,4,5)P3 to PtdIns(3,4)P2 as did recombinant forms of two other 5-phosphatases designated as inositol polyphosphate-5- phosphatase II, and OCRL (the protein that is mutated in oculocerebrorenal syndrome). The inositol polyphosphate-5-phosphatase enzyme family now is represented by at least 9 distinct genes and includes enzymes that fall into 4 subfamilies based on their activities toward various 5-phosphatase substrates.

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A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases. We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II). Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity. Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme. Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P3 when compared to native enzyme [Km = 75 microM, Vm = 8300 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. Mutants W551A and I555A had a Km for Ins(1,4,5)P3 hydrolysis similar to that of the native enzyme (35 microM and 81 microM, respectively), suggesting that these amino acids do not play a role in binding substrate. By contrast, mutant R480A had both increased Km (634 microM) and decreased Vm [855 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. As judged by measurement of Km, mutant R480A retained normal binding of Ins(1,3,4,5)P4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P3 binding than in Ins(1,3,4,5)P4 binding. Mutant I555A bound Ins(1,3,4,5)P4 with 8-fold reduced affinity. These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110). 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2. These results are consistent with a role for these conserved amino acids in substrate binding and catalysis.

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.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, hydrolyzes the 1-phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have used indirect immunofluorescence microscopy, Western blot analysis, and enzyme assays to determine the cellular localization of the enzyme. We find that the enzyme is present, but not exclusively, in the nucleus of Madin-Darby bovine kidney cells, and also in COS-7 and HeLa cells that were transiently transfected with a cDNA encoding bovine inositol polyphosphate 1-phosphatase. DNA synthesis, as measured in COS-7 and HeLa cells transiently over-expressing enzyme, was reduced 50% in cells transfected with wild-type enzyme compared with nontransfected cells or cells transfected with an inactive mutant form of the enzyme. These data demonstrate that this response is mediated by one of the substrates or products of inositol polyphosphate 1-phosphatase. We propose that overexpressed inositol polyphosphate 1-phosphatase degrades a stimulatory inositol phosphate(s) and thereby inhibits DNA synthesis.

In the current study we have measured phosphatidylinositols during the cell cycle. HeLa cells were labeled with [3H]myoinositol to a steady state, synchronized to the G1/S boundary, and the levels of phosphatidylinositol (PtdIns) lipids were measured at various times after release from the block. The levels of total cellular PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 relative to total cellular phospholipid did not vary throughout the cell cycle. We then isolated nuclei from synchronized cells using a non-detergent method and found that the levels of nuclear PtdIns lipids decreased by over 50% at 2 and 4 h after release from the G1/S boundary (S-phase of the cell cycle) and returned to the original levels by 9 h. Separation of individual inositol-containing nuclear lipids showed that PtdIns decreased by 50% while levels of PtdIns(4)P and PtdIns(4,5)P2 decreased by 66%. Levels of the cytoplasmic PtdIns lipids remained constant throughout this period. This experiment indicates that there is specific nuclear. PtdIns turnover that is activated during DNA synthesis.

Inositol polyphosphate 4-phosphatase is a monomeric 110-kDa protein that hydrolyzes two substrates in the inositol phosphate pathway. Inositol 3,4-bisphosphate is converted to inositol 3-phosphate, and inositol 1,3,4-trisphosphate is converted to inositol 1,3-bisphosphate. We have exploited the fact that inositol hexasulfate inhibits the enzyme to devise an affinity elution scheme from a Mono S cation exchange column that resulted in an 11,300-fold purified preparation of rat brain 4-phosphatase. The resulting 4-phosphatase hydrolyzed phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate with a first order rate constant 120-fold greater than that for inositol 3,4-bisphosphate and 900-fold greater than that for inositol 1,3,4-trisphosphate. This is now the third example wherein the same enzyme hydrolyzes both an inositol lipid and its analogous inositol phosphate.

We have investigated a previously little-studied subfamily of related active MTM proteins namely MTMR6, MTMR7, and MTMR8. All three of these proteins partner with the same inactive MTMR9 thus potentially creating competition. We find that complex formation in each case greatly stabilizes all of the proteins. In the absence of complex formation all of the proteins are markedly unstable. We examined the substrate specificity of the proteins and found that the MTMR8/MTMR9 complex prefers PtdIns(3)P as substrate while the MTMR6/MTMR9 complex prefers PtdIns(3,5)P2. In view of this difference the two complexes serve different functions in cells. The MTMR8/MTMR9 complex serves to inhibit autophagy while the MTMR6/MTMR9 complex inhibits apoptosis. We have also created a mouse heterozygous for Mtmr9 that displays a very strong phenotype with small size, ataxia, seizures, unrestrained growth of the teeth, and in most animals premature death. The heterozygous mice express markedly reduced levels of all of these MTM proteins presumably reflecting the instability of the monomeric proteins. This instability results in levels of the proteins that are much less than that expected in heterozygous animals.

In 2005, Herb Tabor called me to ask what I thought should be done to highlight the 100th anniversary of the JBC. I said I really liked the "Reflections" of the old greats of biochemistry. Herb agreed and said, "Someday, when you are older, I will ask you to write one." I thought: "I hope I last that long!" After writing my "Reflections," I feel that my two most significant contributions were delineating the mechanism by which aspirin affects platelet function and defining the scope of inositol signaling reactions. Aspirin acetylates platelet cyclooxygenase for the life of a platelet and acts as an antithrombotic agent at low doses. The laboratory has identified and cloned more than a dozen phosphatases and kinases that modify inositol phosphates and lipids. Mutations in a number of these enzymes cause neurodegenerative diseases in man and animals.

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.

Platelet activation plays an important role in the development and course of cardiovascular disease. It is triggered by the interaction of subendothelial matrix-bound and/or soluble agonists with platelet surface receptors causing a series of morphological and biochemical changes leading to the recruitment of additional platelets and formation of stable platelet aggregates. In addition to events causing initial activation and recruitment of platelets, signaling continues post-aggregation that promotes stability of the thrombus (Brass et al., 2004). Platelet levels of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) increase dramatically in response to agonist stimulation in an aggregation-dependent manner (Kucera et al., 1990; Nolan et al., 1990; Sultan et al., 1990). Furthermore the increase in platelet PtdIns(3,4)P2 occurs late in platelet aggregation and correlates with the irreversible phase of platelet aggregation (Sorisky et al., 1992; Sultan et al., 1991; Trumel et al., 1999), suggesting that PtdIns(3,4)P2 mediates the stabilization of platelet aggregates. However little is known about the regulation of PtdIns(3,4)P2 in platelets. PtdIns(3,4)P2 can be formed by three different routes 1) by direct phosphorylation of PtdIns(4)P by phosphatidylinositol 3-kinase (EC2.7.1.153) (PI 3-K) 2) by PI 3-K using PtdIns(4,5)P2 as a substrate followed by dephosphorylation by a 5-phosphatase (EC3.1.3.56) and, 3) by the action of type I PtdIns(4)P phosphate kinase (EC2.7.1.68) using PI 3-K as substrate (Zhang et al., 1998), shown diagramatically in figure 1. It is well documented that different PI 3-K isoforms play important roles in both early and later stages of platelet aggregation (Jackson et al., 2006). It has been shown that that pharmacologic inhibition of PI 3-K prevents agonist-induced formation of PtdIns(3,4)P2 (Kovacsovics et al., 1995; Schoenwaelder et al., 2007). Furthermore addition of PI 3-K inhibitors after the onset of platelet aggregation induces a decline in PtdIns(3,4)P2 and disaggregation of platelets, supporting a role for PtdIns(3,4)P2 in the stabilization of aggregates. However, platelet agonist-dependent activation of PI 3-K mediates an increase in both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 making approaches using pharmacologic inhibition or genetic disruption of PI 3-K unsuitable to distinguish the contributions of the individual 3-phosphorylated phosphoinositides to platelet signaling. The levels of inositol lipids in cells and their distribution in discrete cellular compartments are regulated by the balance of the enzymatic activities of kinases and phosphatases. Figure 1 Pathways for the formation of PtdIns (3,4)P2. The major route of PtdIns(3,4)P2 hydrolysis is the removal of the D4 phosphate by the enzymes cloned and characterized in our lab (Norris et al., 1997; Norris et al., 1995), inositol polyphosphate 4-phosphatase type I (EC3.1.3.40) and type II (EC3.1.3.66) that are magnesium-independent phosphatases. Over their entire sequence type I and type II 4-phosphatases are 37% identical (Norris et al., 1997). The active site region is more highly conserved, and contains a consensus sequence found in other magnesium-independent phosphatases (Zhang et al., 1994). The consensus sequence CX5 RT/S, is conserved throughout 4-ptases from C. elegans to humans as shown in figure 2. These enzymes do not catalyze the hydrolysis of lipids other than PtdIns(3,4)P2 and therefore provide unique means for the study of this lipid in platelet activation. We have shown that 4-ptase I forms a complex with PI 3-K in platelets which localizes the complex to sites of PtdIns(3,4)P2 production (Munday et al., 1999). Figure 2 Alignment of active sites of 4ptases. We postulate that PtdIns(3,4)P2 is important for platelet function and will study this using a mouse model. We previously showed that an antibody that reacts with 4-phosphatases immunoprecipitates all of the PtdIns(3,4)P2 hydrolyzing activity from human platelets (Munday et al., 1999). An early indication that PtdIns(3,4)P2 was important for platelet function was the work of Norris (Norris et al., 1997). It was shown that calpain caused degradation of recombinant 4-phosphatase I in vitro thereby inactivating it. It was also shown that activation of human platelets with either calcium ionophore or thrombin led to proteolysis of endogenous platelet 4-phosphatase I. If calpeptin, a cell-permeable inhibitor of calpain, was included in these experiments no proteolysis was seen. The levels of PtdIns(3,4)P2 in platelets were lower when calpeptin was included, indicating that 4-phosphatase I was important for controlling the levels of PtdIns(3,4)P2 during platelet activation. A naturally occurring mutation in type I 4-phosphatase is a single nucleotide deletion which is found in the weeble mouse. These animals suffer from severe neurodegeneration and die within the first weeks of life. Therefore such mutant mice cannot be used to study platelet function. We circumvented this problem by creating chimeric mice by bone marrow transplantation of weeble fetal liver cells into lethally irradiated wild type mice. These mice lack 4-phosphatase in bone marrow derived cells including platelets. The mice are viable, but lack platelet 4-phosphatase I.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.

Inositol 2-phosphate (Ins(2)P) has been identified in several cell types. The cellular levels of Ins(2)P appear to be directly correlated with the levels of inositol 1:2-cyclic phosphate (cIns(1:2)P) (Ross, T. S., Wang, F. P., and Majerus, P. W. (1992) J. Biol. Chem. 267, 19919-19923). In this study we have detected an enzyme in extracts from CV-1 cells and rat cerebellum that converts cIns(1:2)P to Ins(2)P and inositol 1-phosphate. This enzyme (designated cyclic hydrolase II) is not the same protein previously designated cIns(1:2)P 2-phosphohydrolase (cyclic hydrolase I). The products, heat inactivation curves, pH optima, and metal dependence of these two activities are different, and the two activities were separated by DEAE and gel filtration chromatography. Mixing of cyclic hydrolase I with cyclic hydrolase II does not effect the activity of either. The Km of the CV-1 cyclic hydrolase II for D-cIns(1:2)P is 10 microM. The enzyme is approximately 55 kDa as estimated by gel filtration analysis in the presence of sodium chloride and 120 kDa in its absence.

PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 C ONVENTIONAL PHOSPHA TIDYLIN OSITOLS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 PHOSPHOLIPASE C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . 227 ACTIVATION Of PHOSPHOLIPASE C BY GaQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . 227 ACTIVATI ON OF PLCy BY TYR OSINE PHOSPHORYLATI ON . . . . . . . . . . . . . . . . . . . . . . . . 229 INOSITOL POLYPHOSPHATE 5-PHOSPHATASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1 INS ( 1 ,4,5)P3 3·KINASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 INOSITOL POLYPHOSPHATE I·PHOSPHATASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 INOSITOL POLYPHOSPHATE 4·PHOSPHATASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 INOSITOL P OLYPHOSPHATE 3·PHOSPHATASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 INOSITOL M ON OPHOSP HATA SE . . . . . . . . . . . .. . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 236 INOSITOL ( 1 :2 CYCLIC) PHOSPHATE 2 PHOSPHOHYDROLASE AND IN OSITOL CYCLIC PHOSPHATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 OTHER ENZYMES OF IN OSITOL PHOSPHATE METAB OLISM . . . . . . . . . . . . . . . . . . . . . . . 238 IN OSITOL PENTAPHOSPHATE AN D HE XAPHOSPHATE METAB OLISM . . . . . . . . . . 238 IN OSITOL PHOSPHATE-BIN DIN G PROTEINS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 PHOSPHATIDYLINOSITOL 3·PHOSPHATE PATHWAy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation. We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries. The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting. The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases. The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast. Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity. PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed. The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.

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 β-galactosidase (βgal) 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 βgal 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 × 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.

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.

We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.

The cDNA that encodes inositol-1,2-cyclic phosphate 2-phosphohydrolase (cyclic hydrolase), an enzyme that converts inositol 1,2-cyclic phosphate (cIns(1,2)P) to inositol 1-phosphate, was expressed in 3T3 cells to investigate the function of inositol cyclic phosphates. Cells with increased cyclic hydrolase activity had lower levels of cIns(1,2)P and grew to a lower density at confluence than control cells. This relationship was strengthened by the demonstration that several cell types with differences in cyclic hydrolase activity had levels of cIns(1,2)P and saturation densities that also correlated inversely with cyclic hydrolase activity. In addition, cyclic hydrolase activity is higher in cells at confluence compared to subconfluence. These results suggest that cellular cIns(1,2)P levels are determined by cyclic hydrolase activity and play a role in the control of cell proliferation.

PtdIns (3, 4, 5) P3 is formed rapidly in NIH-3T3 cells stimulated with platelet derived growth factor (PDGF). We have determined the pathway of formation of this lipid in these cells. Cells were labeled briefly with 32PO4 and then stimulated with PDGF under conditions where the specific activity of each phosphate group determines the order of its addition. The D-5 phosphate of this lipid contained approximately 42% of the total radioactivity present in the molecule, while approximately 32% was in the D-4 position, 25% in the D-3 position, and approximately 2% in the D-1 position. This indicates that PtdIns (3, 4) P2 and not PtdIns (4, 5)P2 is the immediate precursor of PtdIns (3, 4, 5) P3, and defines the pathway of formation of these lipids to be PtdIns (3) P----PtdIns (3, 4) P2----PtdIns (3, 4, 5) P3. This pathway is the same as that in thrombin-stimulated platelets and infers that the pathways are not different in non-growing and proliferating cells.