Identification of lipid inhibitor of mammalian phospholipase D.

Phospholipase D (PLD) is implicated in important cellular processes, such as hormone action, inflammation, secretion, mitogenesis, and neural activity. Recent studies using cell-free systems have shown that the enzyme activity is modulated by both positive and negative regulators. During an attempt to purify PLD from pig colon mucosa, we noted the presence of a PLD inhibitor in the tissue extract. The inhibitor was purified and identified as comprising lysophosphatidylserine, phosphatidylinositol, and lysophosphatidylinositol, of which lysophosphatidylserine was the most potent. These lipids affected all of the PLD isoforms examined, oleate-dependent PLD, ARF-dependent PLD (PLD1a, PLD1b), and phosphatidylinositol 4,5-bisphosphate-dependent PLD (PLD2), in the concentration range of the 1 or 10 microM order. In contrast to lysophosphatidylserine, the diacyl counterpart phosphatidylserine was without effect in the same concentration range. PLD inhibition by lysophosphatidylserine could not be reversed by an increase in the concentration of the substrate phosphatidylcholine or activator phosphatidylinositol 4,5-bisphosphate.

Cloning, differential regulation and tissue distribution of alternatively spliced isoforms of ADP-ribosylation-factor-dependent phospholipase D from rat liver.

An alternatively spliced isoform of ADP-ribosylation-factor-dependent phospholipase D (PLD1) was previously shown to occur in rat C6 cells [Yoshimura, Nakashima, Ohguchi, Sakai, Shinoda, Sakai and Nozawa (1996) Biochem. Biophys. Res. Commun. 225, 494-499] and human HeLa cells [Hammond, Jenco, Nakashima, Cadwallader, Gu, Cook, Nozawa, Prestwich, Frohman and Morris (1997) J. Biol. Chem. 272, 3860-3868]. However, its complete sequence and the enzymological difference between the two PLD1 isoforms were unclear. Here we report the cloning, complete sequence, enzymological properties and tissue distribution of each of the two alternatively spliced PLD1 isoforms, a and b, from rat liver. The major difference between the two isoforms was the deletion of 38 amino acids in the b isoform, but otherwise the two cDNA sequences were 99.9% identical. The a-isoform sequence was 91% identical with the a form of human PLD1, and the 38-amino-acid deletion in the b form occurred at the same site as in the b form of human PLD1. Both of the rat PLD1 isoforms expressed in the fission yeast Schizosaccharomyces pombe were dependent on ADP-ribosylation factor 1 and phosphatidylinositol 4,5-bisphosphate. The a isoform was activated by RhoA in a synergistic manner with ADP-ribosylation factor 1, whereas the b isoform was less responsive to RhoA. Reverse transcription PCR showed that the b form was the predominant PLD1 isoform expressed in rat tissues. The b-form transcript occurred in various rat tissues, including lung, brain, liver, kidney, small intestine and colon, whereas the a-form transcript was only detectable in lung, heart and spleen. Both transcripts were hardly detectable in thymus, stomach, testis and muscle. Thus the two PLD1 isoforms were differently regulated and expressed in rat tissues.

Enhanced levels of oleate-dependent and Arf-dependent phospholipase D isoforms in experimental colon cancer.

Phospholipase D (PLD) (EC 3.1.4.4) is one of the intracellar signal transduction enzymes and plays an important role in a variety of cellular functions. In order to clarify the role of PLD in proliferation and tumorigenesis of colon cancer, we investigated the activities of oleate-dependent and ADP-ribosylation factor (Arf)-dependent types of PLD in experimental colon cancer of the rat. We produced colon cancer in Wistar rats by injecting the carcinogen, dimethylhydrazine dihydrochloride (DMH). The control rats were injected with physiological saline. Mucosal scrapings from the colon were homogenized and centrifuged to obtain the microsomal or membrane fraction. We measured the two types of PLD activities in these fractions using the transphosphatidylation reaction. Both oleate-dependent and Arf-dependent PLD activities were significantly higher in the colon cancer tissue than normal colonic mucosa. The mean specific activity of oleate-dependent PLD) in colon cancers was 1.66 +/- 0.75 (SD) nmol/min/mg whereas the value for normal colonic mucosa was 0.18 +/- 0.09 nmol/min/mg (P < 0.01; Mann-Whitney U-test). On the other hand, the mean specific activity of Arf-dependent PLD in colon cancers was 76.36 +/- 29.37 pmol/min/mg whereas the value for normal colonic mucosa was 19.90 +/- 11.97 pmol/min/mg (P < 0.01; Mann-Whitney U-test). These results suggest that PLD is implicated in the proliferation and tumorigenesis of colon cancer. The present study provides the first evidence for the enhanced levels of two types of PLD in colon cancer and raises the possibility that these PLDs can be used as the potential target for the treatment of colon cancer.

Cloning, expression, and characterization of a novel phospholipase D complementary DNA from rat brain.

Phospholipase D (PLD) is implicated in important cellular processes such as signal transduction, membrane trafficking, and mitosis regulation. Recently, cDNA for human PLD1 (hPLD1) was cloned from HeLa cells (Hammond, S. M., Altshuller, Y. M., Sung, T-C., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). hPLD1 is stimulated by phosphatidylinositol 4,5-bisphosphate and the small GTP-binding protein known as ADP-ribosylation factor 1. Here we report the cloning and characterization of cDNA for a different type of PLD (rat PLD2 (rPLD2)) from rat brain. We synthesized highly degenerate amplimers corresponding to the conserved regions of eukaryote PLDs and performed polymerase chain reaction on a rat brain cDNA library. Using the amplified sequence as the probe, we cloned a rat brain cDNA clone that contained an open reading frame of 933 amino acids with an Mr of 105,992. The deduced amino acid sequence showed significant similarity to hPLD1 with a large deletion in the middle of the sequence. When the sequence was expressed in the fission yeast Schizosaccharomyces pombe, PLD activity was greatly increased. The activity was markedly stimulated by phosphatidylinositol 4, 5-bisphosphate, but not by ADP-ribosylation factor 1 and RhoA. Rat brain cytosol known to stimulate small GTP-binding protein-dependent PLD did not stimulate rPLD2 expressed in S. pombe. The transcript was detected at significant levels in brain, lung, heart, kidney, stomach, small intestine, colon, and testis, but at low levels in thymus, liver, and muscle. Only a negligible level was found in spleen and pancreas. Thus rPLD2 is a novel type of PLD dependent on phosphatidylinositol 4,5-bisphosphate, but not on the small GTP-binding proteins ADP-ribosylation factor 1 and RhoA.

Modulation of the substrate specificity of the mammalian phosphatidylinositol 3-kinase by cholesterol sulfate and sulfatide.

The substrate specificity of the purified, mammalian phosphatidylinositol 3-kinase is subject to modulation by detergents, which are able to switch substrate specificity in vitro in favor of PtdInsP2. This effect of the detergents is due to an activation of the phosphatidylinositol biphosphate 3-kinase activity, while the phosphatidylinositol 3-kinase activity is inhibited. The selective inhibition of the phosphatidylinositol 3-kinase activity (p110 alpha/p85 alpha) is shown here also to be observed by employing cholesterol sulfate or sulfatide at low micromolar concentrations, whereas cholesterol and androsterone sulfate fail to inhibit. These naturally occurring sulfated lipids have at these concentrations no effect on the phosphatidylinositol bisphosphate 3-kinase activity but inhibit the manganese-dependent intrinsic protein kinase activity, thus switching substrate specificity toward the more highly phosphorylated inositol lipids. Cholesterol sulfate and sulfatide inhibit the free catalytic subunit p110 alpha but fail to inhibit the homologous phosphatidylinositol 3-kinase from Saccharomyces cerevisiae (Vps34p), suggesting that these sulfated lipids act specifically on the mammalian phosphatidylinositol 3-kinase. Consistent with this specificity, the regulatory subunit (p85), which is not conserved in the yeast enzyme, is found to play an important role for the affinity of these inhibitors. The implications for the phosphatidylinositol 3-kinase activity in vivo are discussed.

The SNF2/SWI2/GAM1/TYE3/RIC1 gene is involved in the coordinate regulation of phospholipid synthesis in Saccharomyces cerevisiae.

Genes involved in the phospholipid synthesis of Saccharomyces cerevisiae, such as PEM1, PEM2, PSS, and INO1, are coordinately repressed by myo-inositol and choline. In order to investigate this regulation, we transformed wild-type yeast with a PEM1 promoter-lacZ fusion and isolated two mutants, named ric1 and ric2 (regulation by myo-inositol and choline), exhibiting decreased PEM1 expression. The lowered PEM1 expression in the mutants was monitored in colonies in terms of their failure fully to develop blue color on 5-bromo-4-chloro-3-indolyl-beta-galactopyranoside-containing agar. ric1 mutant was auxotrophic for myo-inositol, indicating that INO1 expression was also affected, whereas ric2 mutant required myo-inositol only in the presence of choline. The RIC1 gene was isolated by complementation of the Ino- phenotype of ric1 mutant and its identity was confirmed by genetic cross between the original ric1 mutant and a gene disruptant. The RIC1 gene was sequenced and found to be identical with the previously identified gene, SNF2/SWI2/GAM1/TYE3, which is known to encode a general transcription factor required for the expression of various genes including INO1. Analysis using various lacZ fusion constructs containing promoters for genes in phospholipid synthesis revealed that the expression of myo-inositol-choline-regulated genes, PEM1, PEM2, PSS, CKI, and INO1, was markedly decreased in the snf2/swi2/gam1/tye3/ric1 background, but the expression of a constitutive gene, PIS, was not. We conclude that SNF2/SWI2/GAM1/TYE3/RIC1 is a positive regulatory gene required for the expression of not only INO1 gene, but also of myo-inositol-choline-regulated genes in general.

Cloning and sequence of the SCS3 gene which is required for inositol prototrophy in Saccharomyces cerevisiae.

The SCS3 gene of Saccharomyces cerevisiae was cloned by functional complementation, using a conditional mutant exhibiting myo-inositol auxotrophy in the presence of choline, and sequenced. The sequence contained an open reading frame capable of encoding 380 amino acids with a calculated molecular weight of 42,734. Disruption of the SCS3 locus caused myo-inositol auxotrophy. The gene appeared to be involved in the synthesis of inositol phospholipids from inositol but not in the control of inositol synthesis.

The activation of phosphatidylinositol 3-kinase by Ras.

BACKGROUND: Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined. Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex. We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras. RESULTS: Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression. In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex. In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not. The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins. The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides. Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated. CONCLUSIONS: The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha. This complex can, however, be activated by Ras. We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase.

The mammalian Ptdlns 3-kinase is shown to be inhibited by low nanomolar concentrations of demethoxyviridin, an antifungal agent structurally related to wortmannin. The inhibitory potency of both compounds could be observed in purified Ptdlns 3-kinase whether or not the regulatory subunit (p85 alpha) was present, suggesting that the inhibitors bind to the catalytic subunit (p110) of the Ptdlns 3-kinase. These inhibitors also show similar potency against the intrinsic p85-phosphorylating activity of the p110-kinase. However, the structurally related Ptdlns 3-kinase from Saccharomyces cerevisiae (Vps34p) is not inhibited by either compound. Both inhibitors target the mammalian Ptdlns 3-kinase in vitro and in vivo, implying that these compounds should be useful in suppressing Ptdlns 3-kinase in mammalian systems. The inhibitors did not affect the mammalian Ptdlns 4-kinase, but they are able to inhibit a membrane-associated Ptdlns 4-kinase from Schizosaccharomyces pombe.

Mammalian phosphatidylinositol 3'-kinase induces a lethal phenotype on expression in Schizosaccharomyces pombe; comparison with the VPS34 gene product.

The 110-kDa catalytic subunit of the phosphatidylinositol 3'-kinase (p110) is shown to be expressed in Schizosaccharomyces pombe as a functional protein, as judged by the accumulation of 3'-phosphorylated lipids in vivo and the extraction of 3'-kinase activity in vitro. On expression of p110, the cells fail to grow and lose viability. In contrast, while the Saccharomyces cerevisiae protein Vps34p can be expressed in S. pombe as a functional, extractable, phosphatidylinositol 3-kinase, expression of this protein fails to increase substantially 3'-phosphorylated lipids in vivo and does not induce a phenotype equivalent to that induced by p110. The results indicate that (over-)-accumulation of 3'-phosphorylated inositol lipids in S. pombe causes loss of viability.

Cloning and characterization of the SCS1 gene required for the expression of genes in yeast phospholipid synthesis.

A dominant mutation of Saccharomyces cerevisiae, CSE1, caused a decrease in the expression of the INO1 gene product, inositol-1-phosphate synthase. The residual activity was completely repressed by the addition of choline to the medium. A mutant carrying this mutation could not grow in the presence of choline unless inositol was added to the medium. Here we report a suppressor gene of the CSE1 mutation, SCS1 (suppressor of CSE1), which was cloned by complementation of CSE1 with a wild-type multicopy yeast genomic library. The cloned SCS1 gene contained an open reading frame which encoded 304 amino acid residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with that of the INO2 gene. An scs1/ino2 null mutant constructed by gene replacement was viable, but auxotrophic for inositol and choline, and used for determination of the mRNA levels of various phospholipid-synthesizing enzymes. In agreement with the reported data for ino2 mutants the disruptant showed decreased expression of the INO1 and PSS genes, which are known to be regulated by inositol and choline. In addition, we newly found that the disruption of SCS1/INO2 also caused a decrease in the expression of the CKI, PEM1, and PEM2 genes, which we previously showed to belong to the inositol-choline-regulated gene family. These results confirm and strengthen the conclusion that the SCS1/INO2 gene is required for expression of inositol-choline-regulated genes in phospholipid synthesis.

A dominant mutation that alters the regulation of INO1 expression in Saccharomyces cerevisiae.

A dominant, single nuclear gene mutation, CSE1, caused inositol auxotrophy in yeast cells. The inositol requirement was marked when choline was present in the medium. Inositol-1-phosphate synthase, the regulatory enzyme of inositol synthesis, is repressed by inositol, or more profoundly by a combination of inositol and choline in the wild type. In CSE1, the level of inositol-1-phosphate synthase was low and was greatly repressed on the addition of choline alone. In accordance with this, INO1 mRNA encoding the enzyme was low even under the depressed conditions and was profoundly decreased by choline in CSE1. But in the wild type, the addition of choline alone had little effect. An INO1-lacZ fusion was constructed and the control of the INO1 promoter in CSE1 was studied. lacZ expression was repressed not only by inositol, but also by choline in CSE1, whereas it was repressed by inositol, but only slightly by choline in the wild type. CSE1 was unlinked to the INO1 structural gene. Thus CSE1 was thought to be a regulatory mutation. Furthermore, when the CDP-choline pathway was mutationally blocked, choline did not affect INO1 expression, indicating that the metabolism of choline via the CDP-choline pathway is required for INO1 repression.

Functional analysis of the regulatory region of the yeast phosphatidylserine synthase gene, PSS.

The upstream region of the PSS gene contains three positive cis-acting elements, upstream activation sequences 1 and 2 (UAS1 and UAS2) and a TATA box. The 5' end of UAS1 occurs between positions -239 and -209, and that of UAS2 is between positions -172 and -164. UAS2 contains 5'-TTCACATG-3' as a core sequence at positions -161 to -154. Mutational analysis revealed that this octamer is responsible for the control of PSS expression by inositol and choline. The TATA box is located at positions -112 to -108. In addition, PSS contains a negative cis-acting sequence between UAS2 and the TATA box.

Identification of the upstream activation sequences responsible for the expression and regulation of the PEM1 and PEM2 genes encoding the enzymes of the phosphatidylethanolamine methylation pathway in Saccharomyces cerevisiae.

The yeast phosphatidylethanolamine methylation pathway is encoded by two structural genes, PEM1 and PEM2. The abundance of their transcripts was coordinately repressed by myo-inositol and choline. The most upstream transcriptional start sites for PEM1 and PEM2 were mapped at positions -142 and -42 relative to their first ATG codons, respectively. Promoter deletion analysis defined the 5' boundary of the regulatory region of PEM1 between -336 and -332 and that of PEM2 between -177 and -158. The 38-bp sequence between -336 and -299 from PEM1 and the 48-bp sequence between -177 and -130 from PEM2 conferred regulated transcription upon an upstream-activation-sequence-deficient test gene, CYC1-lacZ. Comparison of these two regions revealed the presence of a common octameric sequence, 5-CATRTGAA-3', which occurred twice in the 38-bp PEM1 regulatory region and once, followed by the 5'-AAACCCACACATG-3' GRFI site, in the 48-bp PEM2 regulatory region. When synthesized chemically and placed in front of CYC1-lacZ, a single copy of CATATGAA directed a rather low level of gene expression, but multiple copies produced high-level expression. In both cases, gene expression was sensitive to myo-inositol and choline. The synthesized GRFI site directed considerable but constitute lacZ expression. When used in conjunction with CATATGAA, synergistic, regulated gene expression was obtained. Hence CATRTGAA was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2. Binding proteins to these sequences were demonstrated by electrophoretic mobility shift assay. Protein binding to CATRTGAA was not competitive with binding to the GRFI sequence, and vice versa. CATRTGAA was also found in the upstream regions of other genes encoding phospholipid-synthesizing enzymes, such as choline kinase, phosphatidylserine synthase, and myo-inositol-1-phosphate synthase, known to be repressed by myo-inositol and choline.

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.