XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair.

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs-c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142-XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1.

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.

The CD40-inducible Bcl-2 family member A1 protects B cells from antigen receptor-mediated apoptosis.

CD40 activation is necessary for thymus-dependent humoral immune responses and rescuing both phenotypically immature WEHI-231 B lymphoma cells from B cell antigen receptor-induced cell death and germinal center B cells from spontaneous apoptosis. As some effects of CD40 are probably mediated by differences in gene expression, cDNA expression arrays and RNase protection assays were used to identify the anti-apoptotic Bcl-2 homolog A1 as a CD40-inducible gene in B cell lines and purified germinal center B cells. Sustained CD40-induced A1 upregulation correlated with CD40-mediated rescue of WEHI-231 cells from anti-IgM-induced apoptosis. Moreover, overexpression of A1 specifically protected WEHI-231 cells from anti-IgM-induced apoptosis but not cell death triggered by certain other stimuli.

Differential role for p38 mitogen-activated protein kinase in regulating CD40-induced gene expression in dendritic cells and B cells.

We investigated whether human monocyte-derived dendritic cells (DCs) differed from tonsillar B cells in the set of cell fate genes they express constitutively and in the way these genes are affected after CD40 ligation. In particular, Bcl-2, TNF receptor-associated factor-2 (TRAF2), and TRAF4 were clearly inducible via CD40 in B cells but not in DCs. DCs, unlike B cells, were induced to increase expression of IL-1beta, IL-1Ra, IL-8, IL-12 p40, RANTES, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 after CD40 ligation. We next tested whether CD40-induced signaling pathways were different in DCs vs B cells. In DCs, as in B cells, CD40 ligation activated p38 mitogen-activated protein kinase (MAPK), its downstream target, MAPKAPK-2, and the c-Jun N-terminal kinase. The p38 MAPK-specific inhibitor, SB203580, blocked CD40-induced MAPKAPK-2 activation, but did not affect activation of c-Jun N-terminal kinase. Furthermore, unlike in B cells, extracellular signal-regulated kinase-1 and -2 were activated after CD40 ligation in DCs. SB203580 strongly blocked CD40-induced IL-12 p40 production in DCs at both mRNA and protein levels, while having minimal effect on CD40-induced expression of the chemokine RANTES. In contrast, no detectable IL-12 p40 protein was secreted in CD40-stimulated B cells. Furthermore, CD40-induced mRNA expression of cellular inhibitor of apoptosis protein-2 was also dependent on the p38 MAPK pathway in DCs and differed compared with that in B cells. In conclusion, CD40 induces distinct programs in DCs and B cells, and the set of p38 MAPK-dependent genes in DCs (IL-12 p40 and cellular inhibitor of apoptosis protein-2) is different from that in B cells (IL-10 and IL-1beta).

Syk and Bruton's tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.

Activation of Akt by multiple stimuli including B cell antigen receptor (BCR) engagement requires phosphatidylinositol 3-kinase and regulates processes including cell survival, proliferation, and metabolism. BCR cross-linking activates three families of non-receptor protein tyrosine kinases (PTKs) and these are transducers of signaling events including phospholipase C and mitogen-activated protein kinase activation; however, the relative roles of PTKs in BCR-mediated Akt activation are unknown. We examined Akt activation in Lyn-, Syk- and Btk-deficient DT40 cells and B cells from Lyn(-/-) mice. BCR-mediated Akt activation required Syk and was partially dependent upon Btk. Increased BCR-induced Akt phosphorylation was observed in Lyn-deficient DT40 cells and Lyn(-/-) mice compared with wild-type cells suggesting that Lyn may negatively regulate Akt function. BCR-induced tyrosine phosphorylation of the phosphatidylinositol 3-kinase catalytic subunit was abolished in Syk-deficient cells consistent with a receptor-proximal role for Syk in BCR-mediated phosphatidylinositol 3-kinase activation; in contrast, it was maintained in Btk-deficient cells, suggesting Btk functions downstream of phosphatidylinositol 3-kinase. Calcium depletion did not influence BCR-induced Akt phosphorylation/activation, showing that neither Syk nor Btk mediates its effects via changes in calcium levels. Thus, BCR-mediated Akt stimulation is regulated by multiple non-receptor PTK families which regulate Akt both proximal and distal to phosphatidylinositol 3-kinase activation.

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes.

We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF-kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways.

Different protein tyrosine kinases are required for B cell antigen receptor-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mitogen-activated protein kinase.

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate-sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.

A comparison of signaling requirements for apoptosis of human B lymphocytes induced by the B cell receptor and CD95/Fas.

To define how the signaling pathways that mediate the B cell receptor (BCR) death pathway differ from those responsible for CD95/Fas-mediated death, we compared the BCR and Fas death pathways in two human B cell lines, B104 and BJAB. Both BCR- and Fas-induced apoptosis are blocked by the peptide cysteine protease inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD (mlz)), demonstrating a common requirement caspase activity. Despite this common characteristic, the ability of actinomycin D and cycloheximide to block BCR-induced apoptosis, but not apoptosis induced by Fas cross-linking, suggests that a major difference between these two pathways is their differential requirements for new gene and protein synthesis. BCR- and Fas-mediated apoptosis are both accompanied by activation of stress-activated protein kinase and p38 mitogen-activated protein kinase (MAPK). Activation of both stress-activated protein kinase and p38 MAPK was inhibited by ZVAD (mlz), suggesting the involvement of caspases. To determine the role of p38 MAPK activation in BCR- and Fas-induced apoptosis, we employed SB203580, a specific inhibitor of p38 MAPK. SB203580 inhibited BCR-induced apoptosis, but not apoptosis induced by cross-linking Fas. Furthermore, both actinomycin D and SB203580 inhibited BCR-induced, but not Fas-induced, activation of caspase. Collectively, these findings establish a role for p38 MAPK in BCR-induced apoptosis both upstream and downstream of caspase activity. The p38 MAPK pathway may function to regulate transcriptional or translational events that are critical for BCR-induced apoptosis.

Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase.

The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the beta-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167].

Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.

Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.

A novel, phospholipase C-independent pathway of inositol 1,4,5-trisphosphate formation in Dictyostelium and rat liver.

In an earlier study a mutant Dictyostelium cell-line (plc-) was constructed in which all phospholipase C activity was disrupted and nonfunctional, yet these cells had nearly normal Ins(1,4,5)P3 levels (Drayer, A.L., Van Der Kaay, J., Mayr, G.W, Van Haastert, P.J.M. (1990) EMBO J. 13, 1601-1609). We have now investigated if these cells have a phospholipase C-independent de novo pathway of Ins(1,4,5)P3 synthesis. We found that homogenates of plc- cells produce Ins(1,4,5)P3 from endogenous precursors. The enzyme activities that performed these reactions were located in the particulate cell fraction, whereas the endogenous substrate was soluble and could be degraded by phytase. We tested various potential inositol polyphosphate precursors and found that the most efficient were Ins(1,3,4,5,6)P5, Ins(1,3,4,5)P4, and Ins(1,4,5,6)P4. The utilization of Ins(1,3,4,5,6)P5, which can be formed independently of phospholipase C by direct phosphorylation of inositol (Stephens, L.R. and Irvine, R.F. (1990) Nature 346, 580-582), provides Dictyostelium with an alternative and novel pathway of de novo Ins(1,4,5)P3 synthesis. We further discovered that Ins(1,3,4,5,6)P5 was converted to Ins(1,4,5)P3 via both Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. In the absence of calcium no Ins(1,4,5)P3 formation could be observed; half-maximal activity was observed at low micromolar calcium concentrations. These reaction steps could also be performed by a single enzyme purified from rat liver, namely, the multiple inositol polyphosphate phosphatase. These data indicate that organisms as diverse as rat and Dictyostelium possess enzyme activities capable of synthesizing the second messengers Ins(1,4,5)P3 and Ins(1,3,4,5)P4 via a novel phospholipase C-independent pathway.

Synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate in vitro and in vivo.

The pathway of synthesis and metabolism of bis-diphosphoinositol tetrakisphosphate (PP-InsP4-PP) was elucidated by high performance liquid chromatography using newly available 3H- and 32P-labeled substrates. Metabolites were also identified by using two purified phosphatases in a structurally diagnostic manner: tobacco "pyrophosphatase" (Shinshi, H., Miwa, M., Kato, K., Noguchi, M. Matsushima, T., and Sugimura, T. (1976) Biochemistry 15, 2185-2190) and rat hepatic multiple inositol polyphosphate phosphatase (MIPP; Craxton, A., Ali, N., and Shears, S. B. (1995) Biochem. J. 305, 491-498). The demonstration that diphosphoinositol polyphosphates were hydrolyzed by MIPP provides new information on its substrate specificity, although MIPP did not metabolize significant amounts of these polyphosphates in either rat liver homogenates or intact AR4-2J cells. In liver homogenates, inositol hexakisphosphate (InsP6) was phosphorylated first to a diphosphoinositol pentakisphosphate (PP-InsP5) and then to PP-InsP4-PP. These kinase reactions were reversed by phosphatases, establishing two coupled substrate cycles. The two dephosphorylations were probably performed by distinct phosphatases that were distinguished by their separate positional specificities, and their different sensitivities to inhibition by F- (IC50 values of 0.03 mM and 1.4 mM against PP-InsP5 and PP-InsP4-PP, respectively). In [3H]inositol-labeled AR4-2J cells, the steady-state levels of PP-[3H]InsP5 and PP-[3H]InsP4-PP were, respectively, 2-3 and 0.6% of the level of [3H]InsP6. The ongoing turnover of these polyphosphates was revealed by treatment of cells with 0.8 mM NaF for 40 min, which reduced levels of [3H]InsP6 by 50%, increased the levels of PP-[3H]InsP5 16-fold, and increased levels of PP-[3H]InsP4-PP 5-fold. A large increase in levels of PP-[3H]InsP5 also occurred in cells treated with 10 mM NaF, but then no significant change to levels of PP-[3H]InsP4-PP were observed; there may be important differences in the control of the turnover of these two compounds.

Comparison of the activities of a multiple inositol polyphosphate phosphatase obtained from several sources: a search for heterogeneity in this enzyme.

A multiple inositol polyphosphate phosphatase (formerly known as inositol 1,3,4,5-tetrakisphosphate 3-phosphatase) was purified approx. 22,000-fold from rat liver. The final preparation migrated on SDS/PAGE as a doublet with a mean apparent molecular mass of 47 kDa. Upon size-exclusion chromatography, the enzyme was eluted with an apparent molecular mass of 36 kDa. This enzyme was approximately evenly distributed between the 'rough' and 'smooth' subfractions of endoplasmic reticulum. There was a 20-fold range of specific activities of this phosphatase in CHAPS-solubilized particulate fractions prepared from the following rat tissues: liver, heart, kidney, testis and brain. However, each of these extracts contained different amounts of endogenous inhibitors of enzyme activity. After removal of these inhibitors by MonoQ anion-exchange chromatography, there was only a 2.5-fold range of specific activities; kidney contained the most and brain contained the least. We prepared and characterized polyclonal antiserum to the hepatic phosphatase, which immunoprecipitated 85-100% of both particulate and soluble phosphatase activities. The antiserum also immunoprecipitated, with equivalent efficacy, CHAPS-solubilized phosphatase activities from heart, kidney, testis, brain and erythrocytes (all prepared from rat). Our data strengthen the case that the function of the mammalian phosphatase is unrelated to the metabolism of Ca(2+)-mobilizing cellular signals. The CHAPS-solubilized phosphatase from turkey erythrocytes was not immunoprecipitated by the polyclonal antiserum, and is therefore an isoform that is structurally distinct, and possibly functionally unique.

Effects of aluminium on the hepatic inositol polyphosphate phosphatase.

There is speculation that some of the toxic effects of Al3+ may originate from it perturbing inositol phosphate/Ca2+ signalling. For example, in permeabilized L1210 mouse lymphoma cells, 10-50 microM Al3+ activated Ins(1,3,4,5)P4-dependent Ca2+ mobilization and Ins(1,3,4,5)P4 3-phosphatase activity [Loomis-Husselbee, Cullen, Irvine and Dawson (1991) Biochem. J. 277, 883-885]. Ins(1,3,4,5)P4 3-phosphatase activity is performed by a multiple inositol polyphosphate phosphatase (MIPP) that also attacks Ins(1,3,4,5,6)P5 and InsP6 [Craxton, Ali and Shears (1995) Biochem. J. 305, 491-498]: 5-50 microM Al3+ increased MIPP activity towards both Ins(1,3,4,5)P4 (by 30%) and Ins(1,3,4,5,6)P5 (by up to 500%), without affecting metabolism of InsP6. Higher concentrations of Al3+ inhibited metabolism of all three substrates, and in the case of InsP6, Al3+ altered the pattern of accumulating products. When 1-50 microM Al3+ was present, InsP6 became a less effective inhibitor of Ins(1,3,4,5)P4 3-phosphatase activity; this effect did not depend on the presence of cellular membranes, contrary to a previous proposal. The latter phenomenon largely explains how, in a cell-free system where Ins(1,3,4,5)P4 3-phosphatase is inhibited by endogenous InsP6, the addition of Al3+ can apparently increase the enzyme activity. However, there was no effect of either 10 or 25 microM Al3+ (in either the presence or absence of apotransferrin) on inositol phosphate profiles in either Jurkat E6-1 lymphoma cells or AR4-2J pancreatoma cells.

Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4.

Osmoregulation, inhibitory neurotransmission and pH balance depend on chloride ion (Cl-) flux. In intestinal epithelial cells, apical Cl- channels control salt and fluid secretion and are, in turn, regulated by agonists acting through cyclic nucleotides and internal calcium ion concentration ([Ca2+]i). Recently, we found that muscarinic pretreatment prevents [Ca2+]i increases from eliciting Cl- secretion in T84 colonic epithelial cells. By studying concomitant inositol phosphate metabolism, we have now identified D-myo-inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4), as the inositol phosphate most likely to mediate this uncoupling. A novel, membrane-permeant ester prepared by total synthesis delivers Ins(3,4,5,6)P4 intracellularly and confirms that this emerging messenger does inhibit Cl- flux resulting from thapsigargin- or histamine-induced [Ca2+]i elevations.

Bovine testis and human erythrocytes contain different subtypes of membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphomonoesterases.

1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].

Inositol 1,4,5,6-tetrakisphosphate is phosphorylated in rat liver by a 3-kinase that is distinct from inositol 1,4,5-trisphosphate 3-kinase.

Liver homogenates phosphorylated inositol 1,4,5,6-tetrakisphosphate exclusively to inositol 1,3,4,5,6-pentakisphosphate. Approximately 30% of this phosphorylating activity was associated with the particulate fraction of the cell, in contrast to the inositol 3,4,5,6-tetrakisphosphate 1-kinase, which was 90% soluble. This soluble 1-kinase activity was resolved from the soluble activity that phosphorylated inositol 1,4,5,6-tetrakisphosphate by anion-exchange chromatography. The two phosphorylating activities were also found to be differentially inhibited by inositol 1,3,4-trisphosphate (IC50 for 3-kinase > 100 microM; IC50 for 1-kinase < 1 microM). Thus, we have demonstrated that inositol 1,4,5,6-tetrakisphosphate is phosphorylated directly by a 3-kinase, and inositol 3,4,5,6-tetrakisphosphate is not an obligatory intermediate, in contrast to one previous model (Oliver, K. G., Putney, J. W., Jr., Obie, J. F., and Shears, S. B. (1992) J. Biol. Chem. 267, 21528-21534). Inositol 1,4,5,6-tetrakisphosphate 3-kinase was inhibited by inositol 1,3,4,6-tetrakisphosphate (IC50, 1 microM). Soluble inositol 1,4,5,6-tetrakisphosphate 3-kinase and inositol 1,4,5-trisphosphate 3-kinase were resolved by anion-exchange chromatography. Furthermore, cDNA clones of two isozymes of inositol 1,4,5-trisphosphate 3-kinase from rat and human brain did not phosphorylate inositol 1,4,5,6-tetrakisphosphate. Thus, these two 3-kinase activities are performed by distinct enzymes.

Hepatic Ins(1,3,4,5)P4 3-phosphatase is compartmentalized inside endoplasmic reticulum.

In pursuit of the physiological role of inositol 1,3,4,5-tetrakisphosphate 3-phosphatase, which also attacks inositol pentakisphosphate and inositol hexakisphosphate with much higher affinity (Nogimori, K., Hughes, P.J., Glennon, M.C., Hodgson, M.E., Putney, J.W., Jr., and Shears, S.B. (1991) J. Biol. Chem. 266, 16499-16506), we have studied the subcellular distribution of the enzyme in liver. Initially, we had to overcome the problem that potent endogenous inhibitor(s) compromise the detection of this enzyme in vitro (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834). We partially purified these inhibitor(s) by anion-exchange chromatography and gel filtration; inhibitory activity co-eluted with standard inositol hexakisphosphate and was depleted by treatment with phytase. Thus, subcellular fractions were pretreated with phytase before assay of 3-phosphatase activity. Our experiments revealed that the hepatic 3-phosphatase was nearly exclusively restricted to the endoplasmic reticulum, and there was little or no activity in either the cytosol, plasma membranes, mitochondria, or nuclei. Detergent treatment of microsomes indicated that there was 93 +/- 2% latency to mannose-6-phosphatase, an intraorganelle enzyme activity (Vanstapel, F., Pua, K., and Blanckaert, N. (1986) Eur. J. Biochem. 156, 73-77). Similar latencies were found for the hydrolysis of inositol 1,3,4,5-tetrakisphosphate (95 +/- 1%), inositol 1,3,4,5,6-pentakisphosphate (94 +/- 1%), and inositol hexakisphosphate (93 +/- 2%). Treatment of microsomes with either sodium carbonate or phosphatidylcholine-specific phospholipase C, to release luminal contents, led to solubilization of approximately 90% of 3-phosphatase activity. Thus, hepatic 3-phosphatase has a highly restricted access to inositol polyphosphates in vivo that needs to be accounted for in the determination of the physiological role of this enzyme.