Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6-pentakisphosphate 3-phosphatase activity.

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 3-phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375-13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)) 3-phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P(5) (K(m)=7.1 microM) and PtdIns(3,4,5)P(3) (K(m)=26 microM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P(5) levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P(5) to inositol 1,4,5,6-tetrakisphosphate is a cell signaling event.

Genetic rationale for microheterogeneity of human diphosphoinositol polyphosphate phosphohydrolase type 2.

Selective expression of enzymes that adjust the intensity of turnover of diphosphoinositolpolyphosphates may regulate vesicle trafficking and DNA repair. For example, the type 2 human diphosphoinositolpolyphosphate phosphohydrolases (hDIPP2alpha and 2beta) are distinguished by a solitary amino-acid residue; the type 2beta isoform contains Gln86 whereas the type 2alpha isoform does not, yet the latter has 2-5 fold more catalytic activity than its beta counterpart (J. Biol.Chem. (2000) 12730). We discovered that both alpha and beta-type mRNAs were co-expressed in clonal cell-lines. We sought a genetic explanation for this microheterogeneity. Two BACs containing distinct, but intronless, hDIPP2beta genes were cloned. Only one of these genes could potentially give rise to our previously characterized hDIPP2beta mRNA; the other gene has several sequence differences and, in any case, is likely a processed pseudogene. These BACS were mapped to 1q12-q21 and 1p12-p13 by FISH. No analogous intronless hDIPP2alpha gene was detected by analysis of 21 individual genomic DNAs. However, sequence analysis of a third hDIPP2 gene (at 12q21) places the Gln86 CAG codon within an AGCAG pentamer, offering adjacent, alternate intronic 3'-boundaries. Thus, 'intron boundary skidding' by spliceosomes provides a mechanism for yielding both hDIPP2alpha and hDIPP2beta mRNAs. Our studies expand the repertoire of molecular mechanisms regulating diphosphoinositolpolyphosphate metabolism and function.

The transcriptional regulator, Arg82, is a hybrid kinase with both monophosphoinositol and diphosphoinositol polyphosphate synthase activity.

The Arg82 gene of Saccharomyces cerevisiae encodes a transcriptional regulator that phosphorylates inositol 1,4,5-trisphosphate [Saiardi et al. (1999) Curr. Biol. 9, 1323-1326]. However, some controversy has surrounded the nature of the reaction products. We now show that Arg82 phosphorylates inositol 1,3,4,5-tetrakisphosphate to inositol pentakisphosphate, which is itself converted to two isomers of diphosphoinositol tetrakisphosphate, one of which has never previously been identified. One of the diphosphoinositol phosphates was further phosphorylated by a yeast cell lysate. We propose that Arg82 is an ancestral precursor of two distinct and specific enzyme families: inositol 1,4,5-trisphosphate kinases and diphosphoinositol polyphosphate synthases.

The inositol hexakisphosphate kinase family. Catalytic flexibility and function in yeast vacuole biogenesis.

Saiardi et al. (Saiardi, A., Erdjument-Bromage, H., Snowman, A., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326) previously described the cloning of a kinase from yeast and two kinases from mammals (types 1 and 2), which phosphorylate inositol hexakisphosphate (InsP(6)) to diphosphoinositol pentakisphosphate, a "high energy" candidate regulator of cellular trafficking. We have now studied the significance of InsP(6) kinase activity in Saccharomyces cerevisiae by disrupting the kinase gene. These ip6kDelta cells grew more slowly, their levels of diphosphoinositol polyphosphates were 60-80% lower than wild-type cells, and the cells contained abnormally small and fragmented vacuoles. Novel activities of the mammalian and yeast InsP(6) kinases were identified; inositol pentakisphosphate (InsP(5)) was phosphorylated to diphosphoinositol tetrakisphosphate (PP-InsP(4)), which was further metabolized to a novel compound, tentatively identified as bis-diphosphoinositol trisphosphate. The latter is a new substrate for human diphosphoinositol polyphosphate phosphohydrolase. Kinetic parameters for the mammalian type 1 kinase indicate that InsP(5) (K(m) = 1.2 micrometer) and InsP(6) (K(m) = 6.7 micrometer) compete for phosphorylation in vivo. This is the first time a PP-InsP(4) synthase has been identified. The mammalian type 2 kinase and the yeast kinase are more specialized for the phosphorylation of InsP(6). Synthesis of the diphosphorylated inositol phosphates is thus revealed to be more complex and interdependent than previously envisaged.

Discovery of molecular and catalytic diversity among human diphosphoinositol-polyphosphate phosphohydrolases. An expanding Nudt family.

The turnover of the "high energy" diphosphoinositol polyphosphates by Ca(2+)- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype human diphosphoinositol-polyphosphate phosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2alpha and hDIPP2beta, distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Gln(86)). Candidate DIPP2alpha and DIPP2beta homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2alpha > hDIPP2beta. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX(2)GX(6)G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3'-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.

Inositol polyphosphate multikinase (ArgRIII) determines nuclear mRNA export in Saccharomyces cerevisiae.

The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity [Saiardi et al. (1999) Curr. Biol. 9, 1323-1326]. To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene. This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular [InsP(3)] 170-fold and decreased [InsP(6)] 100-fold, indicating reduced phosphorylation of InsP(3) to InsP(6). Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP(6). Low levels of InsP(6), and considerable quantities of Ins(1,3,4,5)P(4), were synthesized by an ipmk-independent route. Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism.

Diphosphoinositol polyphosphates: the final frontier for inositide research?

The diphosphoinositol polyphosphates comprise a group of highly phosphorylated compounds which have a rapid rate of metabolic turnover through tightly-regulated kinase/phosphohydrolase substrate cycles. The phosphohydrolases occur as multiple isoforms, the expression of which is apparently carefully controlled. Cellular levels of the diphosphoinositol polyphosphates are regulated by cAMP and cGMP in a protein kinase-independent manner. These inositides can also sense a specific mode of intracellular Ca2+ pool depletion. In this review, we will argue that these are characteristics of highly significant cellular molecules.

The human and rat forms of multiple inositol polyphosphate phosphatase: functional homology with a histidine acid phosphatase up-regulated during endochondral ossification.

We have derived the full-length sequences of the human and rat forms of the multiple inositol polyphosphate phosphatase (MIPP); their structural and functional comparison with a chick histidine acid phosphatase (HiPER1) has revealed new information: (1) MIPP is approximately 50% identical to HiPER1, but the ER-targeting domains are divergent; (2) MIPP appears to share the catalytic requirement of histidine acid phosphatases, namely, a C-terminal His residue remote from the RHGxRxP catalytic motif; (3) rat MIPP mRNA is up-regulated during chondrocyte hypertrophy. The latter observation provides a context for proposing that MIPP may aid bone mineralization and salvage the inositol moiety prior to apoptosis.

A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase.

Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.

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].

Functional consequences of natural sequence variation in the activation domain of HIV-1 Rev.

Initial infection with an attenuated form of human immunodeficiency virus type 1 (HIV-1) may give rise to some of the rare asymptomatic infections that have been observed. Recently, data have been presented suggesting that a persistent mutation in the essential activation domain of the HIV-1 Rev regulatory protein might have contributed to the maintenance of the asymptomatic state in one individual. Here, we have used a range of assays for in vivo Rev function to examine whether natural sequence variation in the normally highly conserved Rev activation domain can indeed affect Rev function. Analysis of five distinct natural sequence variants of the Rev domain demonstrated that each produced a two- to fourfold drop in Rev function when compared to the consensus activation domain sequence A sixth sequence, reported for the MN isolate of HIV-1, proved entirely inactive. However, resequencing of this region of the MN genome revealed that this isolate actually encodes a consensus Rev activation domain. Overall, these data reveal that even natural sequence variation in the essential Rev activation domain can result in significantly reduced Rev function and suggest that isolates containing such sequence variation are likely to replicate less effectively.