Inositol and its derivatives: their evolution and functions

Ins and Ins phospholipids are present in and are made by most Archaea and all eukaryotes. Relatively few bacteria possess Ins phospholipids: and only one major grouping, the Actinobacteria, is known to have evolved multiple functions for Ins derivatives. The Ins phospholipids of all organisms, whether they have diradylglycerol or ceramide backbones, seem to use the same Ins1P headgroup stereochemistry, so they are probably made by evolutionarily conserved pathways. It seems likely that an early member of the Archaea made the first phospholipid with an Ins1P headgroup -maybe three billionyears ago – and that amuchlater archaeal descendentwas the ancestral contributor that brought these molecules into the common ancestor of all eukaryotes – maybe two billionyears ago (Michell, 2007, 2008). It will only be possible to infer the likely details of these processes when we have learned much more about the Ins lipid biochemistry of modern archaeons. All eukaryotes make substantial amounts of PtdIns, both as a ‘bulk’ membrane phospholipid and as the precursor of seven phosphorylated derivatives of PtdIns (the polyphosphoinositides; PPIn) and of the ‘GPI anchors’ of cell surface ectoproteins. PtdIns(4,5)P2 – with its many functions – and its precursor PtdIns4P are found in all in eukaryotes. So are PtdIns3P and PtdIns(3,5)P2, which have ubiquitous roles in the regulation of membrane trafficking events. However, synthesis of and signalling by PtdIns(3,4,5) P 3 appears to be confined to a later-evolved group of eukaryotes.     

Inositol derivatives: evolution and functions

Current research on inositols mainly focuses on myo-inositol (Ins) derivatives in eukaryotic cells, and in particular on the many roles of Ins phospholipids and polyphosphorylated Ins derivatives. However, inositols and their derivatives are more versatile than this--they have acquired diverse functions over the course of evolution. Given the central involvement of primordial bacteria and archaea in the emergence of eukaryotes, what is the status of inositol derivatives in these groups of organisms, and how might inositol, inositol lipids and inositol phosphates have become ubiquitous constituents of eukaryotes? And how, later, might the multifarious functions of inositol derivatives have emerged during eukaryote diversification?     

Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase.

Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5-phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).     

Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4, 5-trisphosphate-receptor protein from brain, is induced by inositol 1,3,4,5-tetrakisphosphate and regulated by a membrane-associated 5-phosphatase.

The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 with high affinity and selectivity. The brain-specific p42IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42IP4 can be translocated from membranes by ligand interaction. p42IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P4. This dissociation is concentration-dependent (> 100 nM), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3, 4,5)P3-containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P4. p42IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P3. Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P4. Thus, Ins(1,3,4,5)P4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P4 comparable to that required for p42IP4 translocation. Thus, in vivo p42IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3.     

Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines.

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.     

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P₃ 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P₃ as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P₃/Ins(1,3,4,5)P₄ 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P₃ 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P₃ 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P₃ as substrate which is depending on the fatty acid composition of the substrate.     

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

Many neutrophil functions are mediated by PtdIns(3,4,5)P3 that exerts its role by mediating protein translocation via binding to their PH-domains. Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) binds the same PH domain, competes for its binding to PtdIns(3,4,5)P3, and thus negatively regulates PtdIns(3,4,5)P3 signaling. In neutrophils, chemoattractant stimulation triggers rapid elevation in Ins(1,3,4,5)P4 level. Depletion of Ins(1,3,4,5)P4 by deleting InsP3KB, the major enzyme producing Ins(1,3,4,5)P4 in neutrophils, augments PtdIns(3,4,5)P3 downstream signals, leading to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. InsP3KB gene is also expressed in hematopoietic stem/progenitor cells. In InsP3KB null mice, the bone marrow granulocyte monocyte progenitor (GMP) population is expanded and the proliferation of GMP cells is accelerated. As results, neutrophil production in the bone marrow is enhanced and peripheral blood neutrophil count is elevated. Ins(1,3,4,5)P4 also plays a role in maintaining neutrophil survival. Depletion of Ins(1,3,4,5)P4 leads to accelerated neutrophil spontaneous death. Finally, InsP3KB and Ins(1,3,4,5)P4 are essential components in bacterial killing by neutrophils. Despite of the augmented neutrophil recruitment, the clearance of bacteria in the InsP3KB knockout mice is significantly impaired. Collectively, these findings establish InsP3KB and its product Ins(1,3,4,5)P4 as essential modulators of neutrophil function and innate immunity.     

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.     

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen.

Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (PtdIns) kinase activity which phosphorylates PtdIns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of PtdIns(3)P and compounds with predicted PtdIns(3,4)P2 and PtdIns(3,4,5)P3 structures. Complex formation between middle T antigen and PtdIns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with PtdIns 3-kinase activates a distinct inositol metabolic pathway.     

PTEN M-CBR3, a versatile and selective regulator of inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Evidence for Ins(1,3,4,5,6)P5 as a proliferative signal

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor is a phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) 3-phosphatase that plays a crucial role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signaling pathway. Although able to metabolize soluble inositol phosphates in vitro, the question of their significance as physiological substrates is unresolved. We show that inositol phosphates are not regulated by wild type PTEN, but that a synthetic mutant, PTEN M-CBR3, previously thought to be inactive toward inositides, can selectively regulate inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5). Transfection of U87-MG cells with PTEN M-CBR3 lowered Ins(1,3,4,5,6)P5 levels by 60% without detectable effect on PtdInsP3. Although PTEN M-CBR3 is a 3-phosphatase, levels of myo-inositol 1,4,5,6-tetrakisphosphate were not increased, whereas myo-inositol 1,3,4,6-tetrakisphospate levels increased by 80%. We have used PTEN M-CBR3 to study the physiological function of Ins(1,3,4,5,6)P5 and have found that Ins(1,3,4,5,6)P5 does not modulate PKB phosphorylation, nor does it regulate clathrin-mediated epidermal growth factor receptor internalization. By contrast, PTEN M-CBR3 expression, and the subsequent lowering of Ins(1,3,4,5,6)P5, are associated with reduced anchorage-independent colony formation and anchorage-dependent proliferation in U87-MG cells. Our results, together with previously published data, suggest that Ins(1,3,4,5,6)P5 has a role in proliferation.     

Interaction of the brain-specific protein p42IP4/centaurin-alpha1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity

The brain-specific protein p42IP4, also called centaurin-alpha1, specifically binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Here, we investigate the interaction of p42IP4/centaurin-alpha1 with nardilysin (NRDc), a member of the M16 family of zinc metalloendopeptidases. Members of this peptidase family exhibit enzymatic activity and also act as receptors for other proteins. We found that p42IP4/centaurin-alpha1 binds specifically to NRDc from rat brain. We further detected that centaurin-alpha2, a protein that is highly homologous to p42IP4/centaurin-alpha1 and expressed ubiquitously, also binds to NRDc. In vivo interaction was demonstrated by co-immunoprecipitation of p42IP4/centaurin-alpha1 with NRDc from rat brain. The acidic domain of NRDc (NRDc-AD), which does not participate in catalysis, is sufficient for the protein interaction with p42IP4. Interestingly, preincubation of p42IP4 with its cognate ligands D-Ins(1,3,4,5)P4 and the lipid diC8PtdIns(3,4,5)P3 negatively modulates the interaction between the two proteins. D-Ins(1,3,4,5)P4 and diC8PtdIns(3,4,5)P3 suppress the interaction with virtually identical concentration dependencies. This inhibition is highly ligand specific. The enantiomer L-Ins(1,3,4,5)P4 is not effective. Similarly, the phosphoinositides diC8PtdIns(3,4)P2, diC8PtdIns(3,5)P2 and diC8PtdIns(4,5)P2 all have no influence on the interaction. Further experiments revealed that endogenous p42IP4 from rat brain binds to glutathione-S-transferase (GST)-NRDc-AD. The proteins dissociate from each other when incubated with D-Ins(1,3,4,5)P4, but not with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In summary, we demonstrate that p42IP4 binds to NRDc via the NRDc-AD, and that this interaction is controlled by the cognate cellular ligands of p42IP4/centaurin-alpha1. Thus, specific ligands of p42IP4 can modulate the recruitment of proteins, which are docked to p42IP4, to specific cellular compartments.     

SHIP-2 and PTEN are expressed and active in vascular smooth muscle cell nuclei,but only SHIP-2 is associated with nuclear speckles

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P3 phosphatases. Therefore, we investigated the dephosphorylation of [32P]PtdIns(3,4,5)P3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P3 phosphatase assays revealed the production of both [32P]PtdIns(3,4)P2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P3 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [32P]PtdIns(3,4,5)P3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P3 into PtdIns(3,4)P2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.     

High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate

The products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes, by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent pro-tein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. Here, we describe the high-resolution crystal structure of the isolated PH domain of PKB(alpha) in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.     

Product-precursor relationships amongst inositol polyphosphates. Incorporation of [32P]Pi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6-pentakisphosphate in intact avian erythrocytes.

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.     

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3. However, addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) which does not stimulate proliferation in macrophages caused a marked and rapid increase in the levels of Ins(1,4,5)P3, inositol bisphosphate and inositol monophosphate, and a decrease in the amount of PtdIns(4,5)P2. FMLP also evoked a rapid increase in intracellular cytosolic Ca2+ levels, as measured with quin 2 the Ca2+-sensitive fluorescent probe, whereas IL-3 and CSF-1 had no such effect. These results suggest that FMLP stimulates the hydrolysis of PtdIns(4,5)P2 to form the second messenger Ins(1,4,5)P3 which acts to increase the levels of cytosolic Ca2+, and that IL-3- and CSF-1-stimulated proliferation in macrophages is not associated with the formation of PtdIns(4,5)P2-derived second messengers.