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.     

Differential association of phosphatidylinositol 3-kinase, SHIP-1, and PTEN with forming phagosomes

In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)] regulate Fcgamma receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P(3) was slightly more abundant than PI(3,4)P(2) at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P(3) necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P(3)-dependent activities necessary for completion of phagocytosis.     

Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.     

Phosphoinositide 3-kinase: A new effector in signal transduction?

Interest in phosphopinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃], respectively. The cellular levels of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked -thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.     

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.     

Pathway for the formation of D-3 phosphate containing inositol phospholipids in intact human platelets

We have identified the structure of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in human platelets. These lipids accounted for less than 2% of the total 32P incorporated into inositol phospholipids in the platelets. All three lipids were labeled in unstimulated platelets, but incorporation of 32P changed rapidly by 15 s after thrombin stimulation, suggesting that they are important in platelet activation. Specific inositol polyphosphate phosphatases were used to both identify the lipid structures and to determine the route of synthesis of these lipids. During 32P labeling and after thrombin stimulation of human platelets, as much as 60% of the total radioactivity present in PtdIns(3,4)P2 was found in the D-4 phosphate and only 35% in the D-3 phosphate indicating that PtdIns(3)P is the precursor of PtdIns(3,4)P2. In addition, the D-5 and D-4 phosphates of PtdIns(3,4,5)P3 each contained 35-40% of the total radioactivity in the molecule compared with only 18-28% in the D-3 position, suggesting that PtdIns(3,4)P2 and not PtdIns(4,5)P2 is the major precursor of this lipid. These results define the predominant pathway for synthesis of these lipids in platelets as PtdIns----PtdIns(3)P----PtdIns(3,4)P2----PtdIns(3,4,5)P3.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.     

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.     

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.     

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.     

Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells

Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4013-4017). In the present study we describe the isolation and characterization of inositol 1:2-cyclic 4-bisphosphate and inositol 1:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through 18O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets loaded with 45Ca2+ and intact Limulus photoreceptors. Both inositol 1:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol 1:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentration-dependent manner. Injection of inositol 1:2-cyclic 4,5-trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol 1:2-cyclic 4,5-trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate that inositol 1:2-cyclic 4,5-trisphosphate may function as a second messenger in stimulated cells.