Identification of PLC210, a Caenorhabditis elegansPhospholipase C, as a Putative Effector of Ras

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematodeCaenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras. Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematodeCaenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras. Ras proteins are small guanine nucleotide-binding proteins that function as molecular switches by cycling between the active GTP-bound state and the inactive GDP-bound state (for a review see Ref. 1Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). They are essential signaling components that regulate a number of biological responses including proliferation and differentiation of mammalian cells, photoreceptor development in flies, vulval development in nematodes, and mating and growth in yeast. Ras exerts its action through association with effector proteins, which involves the interaction with the effector region (amino acid residues 32–40 in human Ha-Ras) of the GTP-bound form of Ras (1Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). In mammalian cells, the GTP-bound Ras interacts directly with a serine/threonine kinase Raf-1, which results in activation of a phosphorylation cascade including Raf-1, MEK, 1The abbreviations used are: MEK, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase; RalGDS, Ral guanine nucleotide dissociation stimulator; PI-PLC, phosphoinositide-specific phospholipase C; PCR, polymerase chain reaction; MBP, maltose-binding protein; GST, glutathione S-transferase; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; GTPγS, guanosine 5′-O-(3-thiotriphosphate); RA, Ras-associating. and extracellular signal-regulated kinase. Recent searches have identified a number of candidates for mammalian Ras effectors other than Raf-1 and its isoforms B-Raf and A-Raf (for a review see Ref. 2Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (276) Google Scholar). All these molecules bound to Ras in a GTP-dependent manner and required the intact effector region of Ras for the interaction. Furthermore, evidence has been presented recently that Ras can activate such putative effectors in two cases: RalGDS, a guanine nucleotide exchange factor for the Ras-like small guanine nucleotide-binding proteins Ral (3Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (300) Google Scholar), and phosphoinositide 3-kinase (4Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (501) Google Scholar). The nematode Caenorhabditis elegans shares with mammals many key signal transduction pathways essential for metazoan cell growth and differentiation. In this organism, Ras appears to be encoded by a single gene, let-60 (5Beitel G.J. Clark S.G. Horvitz H.R. Nature. 1990; 348: 503-509Crossref PubMed Scopus (325) Google Scholar, 6Han M. Sternberg P.W. Cell. 1990; 63: 921-931Abstract Full Text PDF PubMed Scopus (317) Google Scholar). Extensive genetic studies have demonstrated that LET-60 participates in a signal transduction cascade that includes LIN-45 (Raf), MEK-2 (MEK), SUR-1/MPK-1 (extracellular signal-regulated kinase), and other proteins, which are highly homologous to their mammalian counterparts (for a review see Ref. 7Kornfeld K. Trends Genet. 1997; 13: 55-61Abstract Full Text PDF PubMed Scopus (159) Google Scholar).let-60 functions genetically downstream of let-23, a gene whose product resembles a receptor for epidermal growth factor, in post-embryonic induction of the vulva of hermaphrodites (8Aroian R.V. Koga M. Mendel J.E. Ohshima Y. Sternberg P.W. Nature. 1990; 348: 693-699Crossref PubMed Scopus (328) Google Scholar). This pathway is also shown to be involved in male tail differentiation (9Chamberlin H.M. Sternberg P.W. Development. 1994; 120: 2713-2721PubMed Google Scholar). In addition, genetic evidence indicates thatlet-60 acts to some extent downstream of a fibroblast growth factor receptor, the product of the egl-15 gene, in sex myoblast migration (10DeVore D.L. Horvitz H.R. Stern M.J. Cell. 1995; 83: 611-620Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 11Sundaram M. Yochem J. Han M. Development. 1996; 122: 2823-2833PubMed Google Scholar). However, little is known about the Ras-mediated and Raf-independent signal transduction pathway(s) in C. elegans. To search for LET-60 effectors other than LIN-45 Raf, we have carried out a yeast two-hybrid screening for LET-60-binding proteins. The screening has identified a protein structurally related to PI-PLC. Cloning of the full coding sequence of this 210-kDa protein, designated PLC210, revealed its unique features not found in previously identified PI-PLCs. λACT-RB2, a random-primed mixed stage C. elegans cDNA library constructed in λACT that expresses cDNAs as fusions with the GAL4 activation domain, was provided by Dr. Robert Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK). The library was converted to a plasmid form (pACT-RB2) as described (12Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). The LET-60-coding sequence was amplified by PCR from a C. elegans cDNA library (provided by Dr. Yuji Kohara, National Institute of Genetics, Shizuoka, Japan) and subjected to site-directed mutagenesis to introduce an activating mutation, valine for glycine substitution at position 12, using an oligonucleotide 5′-TGTGGTAGTTGGAGATGTAGGAGTT-3′. The resulting LET-60Val-12-coding sequence was cloned into the pAS2–1 (CLONTECH, Palo Alto, CA) for expression as a fusion with the GAL4 DNA-binding domain. The pACT-RB2 library DNA was co-transformed with pAS2–1-LET-60Val-12 into a yeast reporter strain CG-1945 (CLONTECH), and approximately 4 × 106 transformants were plated on yeast synthetic media lacking histidine, leucine, and tryptophan supplemented with 5 mm 3-amino-1,2,4-triazole. After incubation at 30 °C for several days, His+ colonies were picked up and subjected to a β-galactosidase filter assay as described (13Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). Plasmid DNAs were recovered from His+, LacZ+ colonies by transformation into Escherichia coli, and the recovered pACT plasmids were tested for specificity of interaction by standard techniques (13Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). Inserts of the two positive plasmids pACT4-2 and pACT9-1 were characterized by DNA sequencing and found to represent overlapping cDNA segments corresponding to the C-terminal portion of PLC210. The cDNA segment corresponding to the N-terminal portion was isolated by the “spliced leader sequence PCR” (14Krause M. Epstein H.F. Shakes D.C. Caenorhabditis elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego1995: 513-529Google Scholar) using a pair of the 5′-spliced leader 1-specific primer (5′-GGTTTAATTACCCAAGTTTGAG-3′) and a 3′-primer corresponding to the 5′ end of the pACT9-1 cDNA and using the pACT-RB2 library DNA as a template. This method takes advantage of the highly frequent trans-splicing events in C. elegans that generate mRNA species tagged with the spliced leader sequence at its 5′ end (14Krause M. Epstein H.F. Shakes D.C. Caenorhabditis elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego1995: 513-529Google Scholar). The amplified cDNA fragment was characterized by DNA sequencing and used for construction of the composite full-length protein-coding sequence with the 3′ cDNAs from pACT4-2 and pACT9-1. The corresponding genomic sequence was identified by the BLASTN search (15Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71457) Google Scholar) from the C. elegans genome data base (The C. elegans Genome Sequencing Consortium, Genome Sequencing Center, Washington University School of Medicine, St. Louis, MO and the Sanger Center, Wellcome Trust Genome Campus, Cambridge, UK). Preparation of poly(A)+RNA from the mixed stage worms of C. elegans Bristol N2 strain and Northern blot hybridization were performed as described (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The digoxigenin system (Boehringer Mannheim) was used for signal development. A fragment corresponding to the amino acid residues 612–1861 of PLC210 was cloned into pMAL-c (New England Biolabs, Inc., Beverly, MA) for expression as an MBP fusion in E. coli, yielding pMAL-PLC210(612–1861). pGEX-PLC-δ1 (provided by Dr. Tadaomi Takenawa, University of Tokyo, Japan) was used for expression of the full-length rat PLC-δ1 as a GST fusion in E. coli. MBP-PLC210(612–1861) and GST-PLC-δ1 were purified by affinity chromatography on amylose resin or on glutathione-Sepharose, respectively, and their PI-PLC activities were measured in vitro essentially as described previously (17Homma Y. Emori Y. Shears S. Signaling by Inositides. Oxford University Press, Oxford1997: 99-116Google Scholar). Briefly, the fusion proteins were incubated in 50-μl reaction mixtures containing 50 mm2-(N-morpholino)ethanesulfonic acid, pH 6.8, 10 μm Ca2+/EGTA, 100 mm NaCl, 0.2 mg/ml bovine serum albumin, 0.1 mm dithiothreitol, 90 μm 3HPIP2 (20,000 cpm), and 80 μm phosphatidylethanolamine for 30 min at 30 °C. 3HIP3 produced was extracted and quantitated by liquid scintillation counting. Results are presented as the averages from three independent experiments. Various subfragments corresponding to the amino acid residues x to yof PLC210 were generated by restriction endonuclease digestion of the pACT9-1 insert and cloned into pACT, yielding pACT-PLC210(x-y). The same fragments were also inserted into pMAL-c. Interaction of various subfragments of PLC210 with LET-60Val-12 was examined by the yeast two-hybrid assay employing pACT-PLC210(x-y) and pAS2–1-LET-60Val-12. The β-galactosidase activity was measured by blue color development after incubation with 5-bromo-4-chloro-3-indolyl β-D-galactoside as described (13Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar). Interactions of PLC210 with various effector region mutants of human Ha-Ras were examined similarly by employing pACT9-1 and pGBT10-Ha-Ras carrying the mutations (18Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For the in vitroRas-binding assay, MBP-PLC210(x-y) proteins, expressed in E. coli harboring pMAL-PLC210(x-y) plasmids, were attached to amylose resin and examined for association with the posttranslationally modified form of HaRasVal-12, which was purified from Sf9 insect cells infected with a baculovirus expressing it (19Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Crossref PubMed Scopus (127) Google Scholar). A GST fusionSaccharomyces cerevisiae adenylyl cyclase was solubilized from the crude membrane fraction of a yeast strain FS3–1, harboring the plasmids pAD4-GST-CYR1(606–2026) and YEp-HIS3-ADC1-CAP as described previously (20Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (48) Google Scholar). The supernatant (10 μg of protein) after centrifugation at 100,000 × g for 1 h was used for the adenylyl cyclase assay. Measurements of adenylyl cyclase activity dependent on the GTPγS-bound Ha-Ras and of its inhibition by the purified MBP-PLC210(1570–1861) were carried out as described previously (20Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (48) Google Scholar). By the yeast two-hybrid screening using LET-60Val-12 as a bait, we have identified 70 independent partial cDNA clones encoding LIN-45 Raf, RalGDS-like protein, and other proteins. 2M. Shibatohge, K. and T. two clones with overlapping cDNA pACT4-2 and were found to represent the C-terminal portion of a novel protein 1 The full-length protein-coding sequence was constructed from the of pACT9-1 and pACT4-2 and the 5′ portion of the cDNA by the spliced leader sequence PCR and shown to a acid protein 1 Northern blot of from the mixed stage worms a single class of mRNA with the of approximately which with that predicted from the cDNA sequence 1 The search (15Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71457) Google Scholar) of that PLC210 region highly homologous to the PI-PLC proteins and and region homologous to a of guanine nucleotide exchange proteins for by S. cerevisiae 1 of the and domains of PLC210 with those of the homologous proteins, a PI-PLC and bovine PLC-δ1 that amino acid residues for the activity were these proteins, including those for interaction with R. M. Nature. 1996; PubMed Scopus Google Scholar) 1 an MBP fusion these a activity in vitro in the of 10 μm The specific activity of MBP-PLC210(612–1861) was protein, which was with that of protein, in the same assay the other the N-terminal CDC25-like domain is the homologous to the domains of and and three highly the domains of the exchange on Ras-like small proteins McCormick F. Nature. 1993; PubMed Scopus Google Scholar) 1 These suggest that PLC210 be a protein two The corresponding genomic sequence of PLC210 by two and was from the C. elegans Genome Sequencing However, was some between the amino acid sequence of the product predicted by the with the and that from cDNA This was to of the by the To the of PLC210, the insert of pACT9-1 was into two corresponding to residues and residues region was tested for the to LET-60 in the two-hybrid assay. The that the Ras binding activity of PLC210 in residues not in residues residues were expressed as an MBP fusion in E. coli and tested for association with Ha-Ras in vitro MBP-PLC210(1570–1861) bound to the Ha-Ras. A for down the domain of PLC210 from a by and D.R. Trends Biochem. 1996; Full Text PDF PubMed Scopus Google Scholar). They the of a of 100 amino acid residues, the domain, which was of mammalian RalGDS and and identified a of other proteins bearing this domain by searches of data was and was predicted to a of two However, cDNA sequence that PLC210 the C-terminal residues of the downstream domain, it The fragment corresponding to residues was by digestion into two subfragments containing the domain or 1 A and and the subfragments were examined for in vitro association with Ha-Ras containing was of binding Ha-Ras in a GTP-dependent manner with an affinity with that of However, not Ha-Ras at These results that is for Ras This is the of a predicted domain that is shown to as a functional domain. To that PLC210 is a Ras we examined the of the Ras effector region in association with PLC210 by the two experiments. we tested mutations within the effector region of Ha-Ras the association with PLC210. shown in Ha-Ras mutants and to to PLC210. The same Ha-Ras mutants to to a Ras effector in in the two-hybrid assay (18Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, M. S. A. M. S. A. 1993; PubMed Scopus Google Scholar). the other Ha-Ras mutants and which to to Raf-1 (18Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, C. A. A. M. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar), to PLC210. was to to RalGDS and carrying the domains T. E. J. Biol. Chem. 1996; 271: PubMed Scopus Google R. M. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). we tested PLC210 with S. cerevisiae adenylyl cyclase for binding Ras. The yeast adenylyl cyclase is a Ras effector whose of interaction with Ras been characterized the GTP-bound Ras interacts with the domain of adenylyl cyclase through its effector region and directly it in vitro Y. Yamawaki-Kataoka Y. S. T. Kataoka T. S. A. 1990; PubMed Scopus Google Scholar). we shown that Raf-1, and were of this activation of adenylyl cyclase by of Ras T. Wang J. Akasaka K. Okada T. Kataoka T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. T. M. Kariya K. Kataoka T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Kariya K. M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). we tested the of MBP-PLC210(1570–1861) on the activation of adenylyl cyclase The of MBP-PLC210(1570–1861) the that of MBP The results of the two that PLC210 interacts with the effector region of of Ha-Ras effector region mutants with PLC210 and other or in a of PI-PLC is of the responses to various extracellular The the of to generate the and the of from protein kinase C. in and activation of protein kinase C have been in events such as and migration (for a review see Ref. S.G. 1995; PubMed Scopus Google Scholar). By using the yeast two-hybrid we have identified a C. elegans PI-PLC as a LET-60 This PI-PLC, PLC210, bound mammalian Ha-Ras and a Ras as not other small guanine nucleotide-binding proteins such as and by yeast two-hybrid The that PLC210 directly with Ras in a GTP-dependent that this association the intact effector region of and that PLC210 with yeast adenylyl cyclase for binding Ras strongly suggest that PLC210 is a novel Ras we not Ras PLC210. A the GTP-bound Ras was in vitro to MBP-PLC210(612–1861) to any on the could be that Ras can activity of PLC210 in with some factor that was lacking in assay. association with Ras PLC210 to a specific membrane containing is a mammalian of PLC210 In mammalian cells, an in PI-PLC activity with growth has been to activation of an of interacts directly with the and is by phosphorylation S.G. 1995; PubMed Scopus Google Scholar). However, the that the of phosphoinositide in cells was three that in cells a of PI-PLC in these cells PubMed Scopus Google Scholar). of has been shown to S.G. 1990; PubMed Scopus Google Scholar). These are with the that an as species of PI-PLC by Ras some in mammalian cell be by events the transformation such as of to known PI-PLC. In this it be to that PLC210 interacts with Ha-Ras not with these mutants by activities on a activity expressed C. A. A. M. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, T. E. J. Biol. Chem. 1996; 271: PubMed Scopus Google Scholar, R. M. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). This has been for by of the two Ras Raf-1 and RalGDS, Ha-Ras can with Raf-1 not with RalGDS and can with RalGDS not with Raf-1 T. E. J. Biol. Chem. 1996; 271: PubMed Scopus Google Scholar, R. M. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). However, it could also be by a between Raf-1 and Ras effector with a Ras binding to that of In this the mammalian of PLC210, could be a to its with Raf-1 in In a number of that activation of PI-PLC to growth and transformation in cells S.G. 1995; PubMed Scopus Google Scholar). All known can be into three and by the the and the PLC-δ1 on the of and amino acid sequence S.G. S.-H. Lee PubMed Scopus Google Scholar). PLC210 from of these not in its also in its The region N-terminal to the and domains of PLC210 is than those of the other and the unique CDC25-like domain. PLC210 has residues the and which are in from those of and isoforms and of isoforms This region not any known structural such as an of domain found in In with the and the isoforms an additional C-terminal region that is for the specific binding to and activation by the of the C-terminal region of PLC210 is in it has a for Ras the domain. These strongly suggest that PLC210 a of a novel class of PI-PLC. Dr. R. Barstead for Dr. Y. for C. Dr. T. for and Dr. for in the yeast two-hybrid also for and A. and Y. for in of this