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Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor alpha (TNF(alpha))-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFalpha-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFalpha-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFalpha-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis.

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

The protein Gle1 is required for export of mRNAs from the nucleus to the cytoplasm in both lower and higher eukaryotic cells. In human (h) cells, shuttling of hGle1 between the nucleus and cytoplasm is essential for bulk mRNA export. To date, no hGle1-interacting proteins have been reported and the mechanism by which hGle1 interacts with the nuclear pore complex (NPC) and mediates export is unknown. To identify proteins that can interact with hGle1, a genome-wide yeast two-hybrid screen was performed. Three potential hGle1-interacting partners were isolated, including clones encoding the C-terminal region of the NPC protein hNup155. This interaction between hGle1 and full-length hNup155 was confirmed in vitro, and deletion analysis identified the N-terminal 29 residues of hGle1 as the hNup155-binding domain. Experiments in HeLa cells confirmed that the nuclear rim localization of the major hGle1 protein variant (hGle1B) was dependent on the presence of these 29 N-terminal residues. This suggests that this domain of hGle1 is necessary for targeting to the NPC. This work also characterizes the first domain in hNup155, a 177 C-terminal amino acid span that binds to hGle1. The mutual interaction between hGle1 and the symmetrically distributed nuclear pore protein Nup155 suggests a model in which hGle1's association with hNup155 may represent a step in the Gle1-mediated mRNA export pathway.

Tumor necrosis factor receptor 1 (TNF-R1) signaling elicits a wide range of biological responses, including inflammation, proliferation, differentiation, and apoptosis. TNF-R1 activates both caspase-mediated apoptosis and NF-kappaB transcription of anti-apoptotic factors. We now report a link between the TNF-R1 and inositol phosphate signaling pathways. We observed that overexpression of inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) inhibited apoptosis induced by TNFalpha. The anti-apoptotic effect by 5/6-kinase is not attributable to NF-kappaB activation, as no changes were detected in the levels of NF-kappaB DNA binding, IkappaBalpha degradation, or anti-apoptotic factors, such as x-linked inhibitor of apoptosis protein. Decreased expression of 5/6-kinase by RNA interference rendered HeLa cells more susceptible to TNFalpha-induced apoptosis. Overexpression of 5/6-kinase in human embryonic kidney 293 cells inhibited TNFalpha-induced activation of caspases-8, -3, and -9, BID, and poly(ADP-ribose) polymerase. However, 5/6-kinase did not protect against Fas-, etoposide-, or cycloheximide-induced apoptosis. Further, 5/6-kinase protected against apoptosis induced by the overexpression of TNF-R1-associated death domain but not Fas-associated death domain. Therefore, we suggest that 5/6-kinase modifies TNFalpha-induced apoptosis by interfering with the activation of TNF-R1-associated death domain.

The COP9 signalosome (CSN) is a complex of eight proteins first identified as a repressor of plant photomorphogenesis. A protein kinase activity associated with the COP9 signalosome has been reported but not identified; we present evidence for inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) as a protein kinase associated with the COP9 signalosome. We have shown that 5/6-kinase exists in a complex with the eight-component COP9 signalosome both when purified from bovine brain and when transfected into HEK 293 cells. 5/6-kinase phosphorylates the same substrates as those of the COP9 signalosome, including IkappaBalpha, p53, and c-Jun but fails to phosphorylate several other substrates, including c-Jun 1-79, which are not substrates for the COP9-associated kinase. Both the COP9 signalosome- associated kinase and 5/6-kinase are inhibited by curcumin. The association of 5/6-kinase with the COP9 signalosome is through an interaction with CSN1, which immunoprecipitates with 5/6-kinase. In addition, the inositol kinase activity of 5/6-kinase is inhibited when in a complex with CSN1. We propose that 5/6-kinase is the previously described COP9 signalosome-associated kinase.

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.

The enzyme(s) responsible for the production of inositol hexakisphosphate (InsP(6)) in vertebrate cells are unknown. In fungal cells, a 2-kinase designated Ipk1 is responsible for synthesis of InsP(6) by phosphorylation of inositol 1,3,4,5,6-pentakisphosphate (InsP(5)). Based on limited conserved sequence motifs among five Ipk1 proteins from different fungal species, we have identified a human genomic DNA sequence on chromosome 9 that encodes human inositol 1,3,4,5,6-pentakisphosphate 2-kinase (InsP(5) 2-kinase). Recombinant human enzyme was produced in Sf21 cells, purified, and shown to catalyze the synthesis of InsP(6) or phytic acid in vitro. The recombinant protein converted 31 nmol of InsP(5) to InsP(6)/min/mg of protein (V(max)). The Michaelis-Menten constant for InsP(5) was 0.4 microM and for ATP was 21 microM. Saccharomyces cerevisiae lacking IPK1 do not produce InsP(6) and show lethality in combination with a gle1 mutant allele. Here we show that expression of the human InsP(5) 2-kinase in a yeast ipk1 null strain restored the synthesis of InsP(6) and rescued the gle1-2 ipk1-4 lethal phenotype. Northern analysis on human tissues showed expression of the human InsP(5) 2-kinase mRNA predominantly in brain, heart, placenta, and testis. The isolation of the gene responsible for InsP(6) synthesis in mammalian cells will allow for further studies of the InsP(6) signaling functions.

Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells.

Phosphorylation of inositol 1,3,4-trisphosphate by inositol 1,3,4-trisphosphate 5/6-kinase is the first committed step in the formation of higher phosphorylated forms of inositol. We have shown that the eight proteins called the COP9 signalosome complex copurify with calf brain 5/6-kinase. Because the complex has been shown to phosphorylate c-Jun in vitro, we tested both the complex and 5/6-kinase and found that both are able to phosphorylate c-Jun and ATF-2 on serine/threonine residues. These findings establish a link between two major signal transduction systems: the inositol phosphates and the stress response system.

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.

The protein product of the gene that when mutated is responsible for Lowe syndrome, or oculocerebrorenal syndrome (OCRL), is an inositol polyphosphate 5-phosphatase. It has a marked preference for phosphatidylinositol 4,5-bisphosphate although it hydrolyzes all four of the known inositol polyphosphate 5-phosphatase substrates: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The enzyme activity of this protein is determined by a region of 672 out of a total of 970 amino acids that is homologous to inositol polyphosphate 5-phosphatase II. Cell lines from kidney proximal tubules of a patient with Lowe syndrome and a normal individual were used to study the function of OCRL. The cells from the Lowe syndrome patient lack OCRL protein. OCRL is the major phosphatidylinositol 4,5-bisphosphate 5-phosphatase in these cells. As a result, these cells accumulate phosphatidylinositol 4,5-bisphosphate even though at least four other inositol polyphosphate 5-phosphatase isozymes are present in these cells. OCRL is associated with lysosomal membranes in control proximal tubule cell lines suggesting that OCRL may function in lysosomal membrane trafficking by regulating the specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes.

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

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3, 4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.

Inositol polyphosphate 4-phosphatase (4-phosphatase), an enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, was shown to be a substrate for the calcium-dependent protease calpain in vitro and in stimulated human platelets. Stimulation of platelets with the calcium ionophore, A23187, resulted in complete proteolysis of 4-phosphatase and a 75% reduction in enzyme activity. Thrombin stimulation of platelets resulted in partial proteolysis of 4-phosphatase and a 41% reduction in enzyme activity (n = 8, range of 36-51%). In addition, preincubation with the calpain inhibitor, calpeptin, suppressed the accumulation of phosphatidylinositol 3, 4-bisphosphate in thrombin-stimulated platelets by 36% (n = 2, range = 35-37%). These data suggest that the calpain-mediated inhibition of 4-phosphatase is involved in the phosphatidylinositol 3, 4-bisphosphate accumulation in thrombin-stimulated platelets.

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

Pleckstrin is the major substrate phosphorylated on serine and threonine in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a complex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). This enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products mobilize intracellular calcium. Pleckstrin co-immunoprecipitates with 5-phosphatase I in homogenates of platelets. Platelet homogenates fractionated by anion exchange chromatography show co-elution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin have 7-fold greater 5-phosphatase activity than those containing unphosphorylated pleckstrin. Mixing experiments with recombinant 5-phosphatase I and pleckstrin in vitro show that they form a stoichiometric complex. A mutant form of pleckstrin, in which the serine and threonine residues that are phosphorylated by protein kinase C are substituted with glutamic acid (pseudophosphorylated pleckstrin), activates recombinant 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin does not stimulate the enzyme. Thus pleckstrin functions to terminate calcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.

To gain insight into the intracellular signaling cascades that are activated by the binding of interleukin-3 (IL-3) to its target cells, we have embarked on the identification of proteins that are associated with the IL-3 receptor (IL-3R). In a previous study we reported that a 110-kDa serine/threonine protein kinase is constitutively associated with the IL-3R and activated following IL-3 stimulation. We now report that a phosphatidylinositol-3,4, 5-trisphosphate (PtdIns-3,4,5-P3) 5-phosphatase (5-ptase) is also constitutively associated with the IL-3R. This 5-ptase is magnesium-dependent and removes the 5-position phosphate from PtdIns-3,4,5-P3 but does not metabolize PtdIns-4,5-P2, inositol (Ins)-1,3,4,5-P4, or Ins-1,4,5-P3. This substrate specificity distinguishes it from any previously characterized 5-ptase. Interestingly, it may be bound indirectly via phosphatidylinositol 3-kinase (PI 3-kinase), another enzyme that is constitutively bound to the IL-3R. However, unlike PI 3-kinase which becomes activated following IL-3 stimulation, this receptor-associated 5-ptase activity does not increase following IL-3 stimulation, and its primary function may be to keep the principal in vivo product of PI 3-kinase, PtdIns-3,4,5-P3, at low levels in unstimulated cells, to terminate the PI 3-kinase signal following IL-3 stimulation or to metabolize PtdIns-3,4,5-P3 to a metabolically active second messenger, i.e. PtdIns-3,4-P2.

We previously cloned a cDNA encoding a protein tyrosine phosphatase (PTP) containing sequence homology to protein 4.1, designated PTPMEG. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. We prepared recombinant protein in Sf9 and COS-7 cells to further characterize it. The protein was phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation were all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains. The recombinant protein was cleaved by trypsin and calpain in this region and thereby activated 4-8-fold as assayed using Raytide as substrate. We immunoprecipitated the protein from human platelets with both amino- and carboxyl-terminal antipeptide antibodies to assess the state of the enzyme in these cells. The full-length molecule was found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contained proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocked the agonist-induced proteolysis.

Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.

Inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and phosphatidylinositol 3,4-bisphosphate. The amino acid sequences of tryptic and CNBr peptides of the enzyme isolated from rat brain were determined. Degenerate oligonucleotide primers based on this sequence were used to amplify a 74-base pair polymerase chain reaction product. This product was used to isolate a 5607-base pair composite cDNA, which had an open reading frame encoding a protein with 939 amino acids with a predicted molecular mass of 105,588 Da. The rat brain polymerase chain reaction product was used as a probe to isolate a human brain cDNA that predicts a protein with 938 amino acids and a molecular mass of 105,710 Da. Remarkably, the human and rat proteins were 97% identical. Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the 4-phosphatase. Northern blot hybridization indicates that the 4-phosphatase is widely expressed in rat tissues with the highest levels of expression occurring in brain, heart, and skeletal muscle. Polyclonal antiserum directed against the carboxyl terminus of the 4-phosphatase immunoprecipitated > 95% of the 4-phosphatase activity in crude homogenates of rat brain, heart, skeletal muscle, and spleen, suggesting that this enzyme accounts for the 4-phosphate activity present in rat tissues. This antiserum also immunoprecipitated the 4-phosphatase from human platelet sonicates.

We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins(1,4,5)P3 to Ins(1,4)P2 and the phospholipid PtdIns(4,5)P2 to PtdIns(4)P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [3H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns(4,5)P2 is reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.

We have isolated an inositol hexakisphosphate binding protein from rat brain by affinity elution chromatography from Mono S cation exchange resin using 0.1 mM inositol hexakisphosphate (InsP6). The amino acid sequences of six tryptic peptides from the protein were identical to the sequences predicted from the cDNA encoding a previously isolated protein designated as AP-3 or AP180. This protein is localized in nerve endings and promotes assembly of clathrin into coated vesicles. The isolated protein-bound InsP6 with a dissociation constant of 1.2 microM and a stoichiometry of 0.9 mol of InsP6 bound/mol of AP-3. Recombinant AP-3 expressed in Escherichia coli also bound InsP6 with a similar affinity. InsP6 inhibited clathrin cage assembly mediated by AP-3, in an in vitro assay, but had little effect AP-3 binding to preformed cages. We speculate that InsP6 and perhaps highly phosphorylated inositol lipids may play a role in coated vesicle formation.

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

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

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

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

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

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

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

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

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

Glycerophosphoinositol (GroPIns) is a major inositol phosphate in many cell types. In this study we have determined the optimal conditions (pH 8.0 and 0.5 mM MnCl2) for the metabolism of this molecule in an extract from human placenta, and we show that the major product is inositol (1)-phosphate (Ins(1)P). The enzyme activity that catalyzes this reaction is contained in the same protein designated previously as inositol-(1,2)-cyclic-phosphate 2-inositolphosphohydrolase (cyclic hydrolase), a phosphodiesterase that catalyzes the conversion of inositol-(1,2)-cyclic phosphate (cIns(1,2)P) to Ins(1)P. In addition, the enzyme also catalyzes the production of Ins(1)P from inositol (1)-methylphosphate. All of these substrates, (cIns(1,2)P, GroPIns, and inositol (1)-methylphosphate), contain a phosphodiester bond at the 1-position of the inositol ring. Additional phosphate groups on the 4- or 5-positions of the inositol ring prevent hydrolysis by cyclic hydrolase. The Km of the enzyme for GroPIns is 0.67 mM, and the Vm is 5 mumol/min/mg of protein. GroPIns competitively inhibits cIns(1,2)P hydrolysis with a Ki equal to its Km as a substrate. Hydrolysis of GroPIns and cIns(1,2)P is stimulated by MnCl2, phosphatidylserine, and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). However, whereas cIns(1,2)P hydrolysis is increased 5-8-fold by phosphatidylserine and EGTA only a 2-fold increase of GroPIns hydrolysis occurs under the same conditions. Hydrolysis of both GroPIns and cIns(1,2)P is inhibited by Ins(2)P; the ID50 values are 12 and 1 microM, respectively. There are significant quantities of GroPIns and Ins(2)P in 3T3 cells, indicating that these compounds that alter cIns(1,2)P hydrolase activity may modulate intracellular levels of cIns(1,2)P. Finally, we present evidence suggesting that the substrate specificity of this enzyme is altered during cell transformation.

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.

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.

Protein C, a vitamin K-dependent protein, circulates in plasma as an inactive precursor. Once activated, it possesses potent anticoagulant activity through the inactivation of factors Va and VIIIa. Thrombin, the only known physiologic activator of this protein, is catalytically inefficient. Thrombomodulin, a protein purified from rabbit lungs, has been reported to enhance protein C activation by thrombin. We have previously demonstrated that factor Va, a substrate for activated protein C, is also a thrombin cofactor in the activation of protein C (Salem, H.H., Broze, G.J., Miletich, J. P., and Majerus, P.W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1584-1588). When factor Va is fractionated to its individual components, only the light chain (Mr 78,000) has thrombin cofactor activity. Although factor Va and thrombomodulin can both stimulate thrombin-catalyzed protein C activation, the physiological relationship between these two proteins remains to be determined.

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).

Arachidonoyl-CoA synthetase was solubilized from a particulate fraction of calf brain and human platelets using 1% Nonidet P-40 and 10 mM EDTA. Arachidonoyl-CoA synthetase from both preparations was separated from nonspecific (long chain) acyl-CoA synthetase (EC 6.2.1.3) by chromatography on hydroxylapatite. To further substantiate that the two acyl-CoA synthetases are distinct proteins, we solubilized enzyme from a mutant cell line lacking arachidonoyl-CoA synthetase and from the parent cell line from which it was derived. These preparations were chromatographed on hydroxylapatite, and the mutant showed an absence of the peak identified as arachidonoyl-CoA synthetase in the parent while retaining the peak of nonspecific acyl-CoA synthetase activity. We have also determined the levels of arachidonoyl and nonspecific acyl-CoA synthetase in 13 different human cells and tissues. Arachidonoyl-CoA synthetase is widely distributed and is present in significantly lower concentrations than nonspecific acyl-CoA synthetase only in adipose tissue and liver.

Sheep seminal vesicles contain two immunologically distinct phospholipase C (PLC) enzymes that can hydrolyze phosphatidylinositol (PI) (Hofmann, S.L., and Majerus, P.W. (1982) J. Biol. Chem. 257, 6461-6469). One of these enzymes (PLC-I) has been purified to homogeneity; the second (PLC-II) has been purified 2600-fold from a crude extract of seminal vesicles. In the present study we have compared the ability of these purified enzymes to hydrolyze PI, phosphatidylinositol 4-phosphate (PI-4-P), and phosphatidylinositol 4,5-diphosphate (PI-4,5-P2). Using radiolabeled substrates in small unilamellar phospholipid vesicles of defined composition, the two enzymes were found to hydrolyze all three of the phosphoinositides. Hydrolysis of all three phosphoinositides by both enzymes was stimulated by Ca2+; however, in the presence of EGTA only the polyphosphoinositides were hydrolyzed. The two enzymes displayed substrate affinities in the order PI greater than PI-4-P greater than PI-4,5-P2, and maximum hydrolysis rates in the order PI-4,5-P2 greater than PI-4-P greater than PI. When present in the same vesicles, PI and the polyphosphoinositides competed for a limiting amount of either enzyme. Inclusion of phosphatidylcholine into vesicles containing the phosphoinositides resulted in greater inhibition of PI hydrolysis than polyphosphoinositide hydrolysis. When all three phosphoinositides were present in vesicles mimicking the cytoplasmic leaflet of cell membranes, there was preferential hydrolysis of the polyphosphoinositides over PI. We conclude that a single phospholipase C can account for the hydrolysis of all three phosphoinositides seen during agonist-induced stimulation of secretory cells. The cytoplasmic Ca2+ concentration and phospholipid composition of the membrane, however, may influence the relative rate of hydrolysis of the three phosphoinositides.

Protein C, a plasma protein, is activated by thrombin to a protease (protein Ca) that functions as a physiological anticoagulant. We have isolated thrombomodulin, a cofactor required for the rapid activation of protein C, from human placenta. The purification to near homogeneity was achieved using a crude Triton-solubilized protein fraction from a placental particulate fraction as starting material. Chromatography on DEAE-Sepharose removed 95% of the protein and achieved a 3-fold purification. Thrombomodulin was then isolated by affinity chromatography on a column of thrombin-Sepharose wherein the thrombin had been previously inactivated with diisopropyl fluorophosphate. The final preparation was purified 7,900-fold over the membrane extract with a yield of 7%. We obtained 0.88 mg of thrombomodulin from 100 g of membrane extract derived from 5 kg of placenta. The protein was nearly homogeneous as judged by electrophoresis on 10% acrylamide sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol with an apparent Mr = 105,000. Western blot analysis without 2-mercaptoethanol gave an apparent Mr = 75,000. The protein stimulated the rate of protein C activation by thrombin 800-fold to 10 mol of Ca formed/min/mol of thrombin. Thrombin and thrombomodulin appear to form a 1:1 stoichiometric complex as judged from experiments where we measured the effect of varying the concentration of thrombomodulin with respect to thrombin and the converse, on rates of protein C activation. An antibody directed against rabbit lung thrombomodulin inhibited the human placenta protein by 66%, and the amino acid composition of the proteins from the two species was similar indicating that the proteins are closely related. The apparent Michaelis constant of the thrombin-thrombomodulin complex for protein C is 9.8 microM. The protein C activation reaction requires calcium ions and is maximal at 1 mM Ca2+; higher concentrations inhibited the reaction. Coagulation factor Va and factor Va light chain both stimulate the activity of human thrombomodulin 2- to 3-fold.

The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide-dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation.

The level of phosphatidylinositol (PI) in human platelets falls abruptly when they are stimulated with thrombin and then returns to baseline within 15 min. PI is enriched in arachidonate and our recently proposed pathway for arachidonate release (Bell, R. L., Kennerly, D. A., Stanford, N., and Majerus, P. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3238-3241) provides a link between the unique fatty acid composition of PI and the increased turnover of PI seen in many secretory tissues when they are stimulated. We measured the fatty acid composition of PI at time points following stimulation with thrombin as a means of defining the route of PI resynthesis which occurs, and the mechanism for the arachidonate enrichment. In resting platelets, the fatty acid content (per cent of total fatty acid contributed by each) of PI is: palmitate, 5.1; stearate, 39.9; oleate, 8.6; linoleate, 5.6; and arachidonate, 40.8. At 30 s after thrombin treatment, when the mass of PI has fallen from 18.7 +/- 2.3 to 12.5 +/- 2.4 nmol/10(9) platelets, there are no significant changes in per cent fatty acid composition. However, at 8 min, when PI has returned to 16.8 +/- 2.9 nmol/10(9), the per cent fatty acid composition is: palmitate, 7.8 (p > 0.05); stearate, 34.0 (p < 0.005); oleate, 10.7 (p < 0.05); linoleate, 16.1 (p < 0.001); and arachidonate, 31.3 (p < 0.05). The altered fatty acid composition excludes the "Pi cycle" as the mechanism for resynthesis as it would result in an unchanged pattern. The evidence suggests that PI is synthesized without a specific fatty acid composition, and then undergoes a deacylation-reacylation cycle to result in the unique 1-stearyl-2-arachidonyl pattern.

Platelets contain small amounts of a variety of free fatty acids but essentially no free arachidonate. When free fatty acids are incubated with platelets, there is preferential incorporation of arachidonic acid and 8,-11,14-eicosatrienoic acid compared to other fatty acids. We now explain these findings by the discovery that platelets contain two long chain acyl-CoA synthetases. One shows activity with a range of different fatty acids, similar to long chain acyl-CoA synthetases studied previously. A crude platelet membrane preparation contains this enzyme that catalyzes the formation of 0.75 nmol of oleoyl-CoA/min/10(9) platelets. The other enzyme is specific for the prostaglandin precursors arachidonic acid and 8,11,14-eicosatrienoic acid. Based on the ability of fatty acids to inhibit arachidonate and 8,11,14-eicosatrienoate activation, we conclude that other fatty acids including linoleic, 5,8,11-eicosatrienoic, and oleic acids are not substrates for the enzyme. Platelet membranes catalyze formation of 2.9 nmol of arachidonoyl-CoA/min/10(9) platelets and 2.5 nmol of 8,11,14-eicosatrienoyl-CoA/min/10(9) platelets. Arachidonoyl-CoA synthetase has optimal activity at pH 8 and requires ATP (Km = 0.5 mM), Mg2+ (Km = 2.5 mM), CoA (Km = 0.13 mM), and arachidonic acid (Km = 0.03 mM). We propose that the arachidonate-specific acyl-CoA synthetase may control the level of free arachidonic acid in platelets, limiting prostaglandin synthesis by the unstimulated cell and capturing free arachidonate from extracellular sources.