Identification of PLC210, a Caenorhabditis elegans Phospholipase C, as a Putative Effector of Ras*

Mitsushige Shibatohge, Ken-ichi Kariya, Yanhong Liao, Chang-Deng Hu, Yasuhiro Watari, Masahiro Goshima, Fumi Shima, and Tohru KataokaDagger

From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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. 1). 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 (1). 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,1 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. 2). 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 (3), and phosphoinositide 3-kinase (4).

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 (5, 6). 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. 7). 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 (8). This pathway is also shown to be involved in male tail differentiation (9). In addition, genetic evidence indicates that let-60 acts to some extent downstream of a fibroblast growth factor receptor, the product of the egl-15 gene, in sex myoblast migration (10, 11). 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Two-hybrid Screening-- lambda ACT-RB2, a random-primed mixed stage C. elegans cDNA library constructed in lambda ACT 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 (12). 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 beta -galactosidase filter assay as described (13). 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 (13).

Cloning of PLC210 cDNA-- 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" (14) 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 (14). 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 (15) 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).

Northern Blot Analysis-- Preparation of poly(A)+ RNA from the mixed stage worms of C. elegans Bristol N2 strain and Northern blot hybridization were performed as described (16). The digoxigenin system (Boehringer Mannheim) was used for signal development.

Assay for Phospholipase C Activity-- 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-delta 1 (provided by Dr. Tadaomi Takenawa, University of Tokyo, Japan) was used for expression of the full-length rat PLC-delta 1 as a GST fusion in E. coli. MBP-PLC210(612-1861) and GST-PLC-delta 1 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 (17). Briefly, the fusion proteins were incubated in 50-µl reaction mixtures containing 50 mM 2-(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 [3H]PIP2 (20,000 cpm), and 80 µM phosphatidylethanolamine for 30 min at 30 °C. [3H]IP3 produced was extracted and quantitated by liquid scintillation counting. Results are presented as the averages from three independent experiments.

Two-hybrid and in Vitro Binding Assays-- Various subfragments corresponding to the amino acid residues x to y of 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 beta -galactosidase activity was measured by blue color development after incubation with 5-bromo-4-chloro-3-indolyl beta -D-galactoside as described (13). 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 (18). For the in vitro Ras-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 (19).

Adenylyl Cyclase Inhibition Assay-- A GST fusion Saccharomyces 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 (20). 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 GTPgamma S-bound Ha-Ras and of its inhibition by the purified MBP-PLC210(1570-1861) were carried out as described previously (20).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of PLC210, a Novel Phophoinositide-specific Phopholipase C-- 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.2 Among them, two clones with overlapping cDNA inserts, pACT4-2 and pACT9-1, were found to represent the C-terminal portion of a novel Ras-binding protein (Fig. 1A). The full-length protein-coding sequence was constructed from the inserts of pACT9-1 and pACT4-2 and the 5' portion of the cDNA obtained by the spliced leader sequence PCR and shown to encode a 1898-amino acid residue protein (Fig. 1B). Northern blot analysis of poly(A)+ RNA from the mixed stage worms detected a single class of mRNA with the size of approximately 6.5 kilobases, which coincided with that predicted from the cDNA sequence (Fig. 1C). The BLASTP search (15) of GenBankTM entries indicated that PLC210 contained one region highly homologous to the PI-PLC family proteins (the X, Y and C2 domains) and another region homologous to a family of guanine nucleotide exchange proteins for Ras, represented by S. cerevisiae CDC25 (Fig. 1A). Comparison of the X, Y, and C2 domains of PLC210 with those of the most homologous proteins, a Drosophila melanogaster PI-PLC (accession number P25455) and bovine PLC-delta 1 (P10895), indicated that critical amino acid residues for the catalytic activity were conserved among these proteins, including those necessary for Ca2+-dependent interaction with phosphoinositides (21) (Fig. 1D). Indeed, an MBP fusion polypeptide encompassing these domains, MBP-PLC210(612-1861), exhibited a hydrolyzing activity toward PIP2 in vitro in the presence of 10 µM Ca2+. The observed specific activity of MBP-PLC210(612-1861) was 5.3 nmol/min/mg protein, which was comparable with that of GST-PLC-delta 1, 9.1 nmol/min/mg protein, obtained in the same assay condition.


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Fig. 1.   Structural features of PLC210. A, shown is the schematic representation of various functional domains in PLC210. CDC25, CDC25-like domain; X, X domain; Y, Y domain; C2, C2 domain; RA1 and RA2, the upstream and downstream RA domains. The ranges covered by pACT4-2 and pACT9-1 are indicated below. The upper line represents the HindIII fragment used as a probe in the Northern blot analysis. The region expressed as MBP-PLC210(612-1861) is also indicated. B, the complete amino acid sequence of PLC210 deduced from the nucleotide sequence of the cloned cDNA is shown by one-letter codes. C, poly(A)+ RNA (2 µg) isolated from the mixed stage worms (Ce) was subjected to Northern blot analysis with a digoxigenin-labeled 1.1-kilobase HindIII fragment of the PLC210 cDNA as a probe. Included are digoxigenin-labeled RNA molecular weight markers (M) (Boehringer Mannheim). D, optimal alignments of the catalytic and C2 domains of PLC210 and related proteins were produced with a computer program GENETYX-MAC (Software Development Co., Japan) and adjusted manually. Numbers beside the margins represent amino acid positions within the respective proteins. Identical residues are in bold type. The positions of residues important for the binding of inositol trisphosphate and Ca2+ in the catalytic domain as well as putative Ca2+ ligands in the C2 domain (21) are indicated by asterisks. Ce, C. elegans PLC 210; Dm, D. melanogaster PLC; b, bovine PLC-delta 1. E, optimal alignments of the CDC25-like domain of PLC210 (Ce) with those of D. melanogaster Sos (Dm) and mouse Sos2 (m) were produced as in D. Three regions conserved among the catalytic domains of Ras GDP/GTP exchange factors (22) are boxed. Residues homologous to the consensus sequences shown below are represented by italic letters.

On the other hand, the N-terminal CDC25-like domain is the most homologous to the catalytic domains of mouse Sos2 (Z11664) and D. melanogaster Sos (M3931) and contained three regions highly conserved among the catalytic domains of the GDP/GTP exchange factors acting on Ras-like small GTP-binding proteins (22) (Fig. 1E). These observations suggest that PLC210 may be a bifunctional protein exerting two distinct catalytic activities.

The corresponding genomic sequence of PLC210 covered by two contiguous cosmid clones, F31B12 and T05A10, was available from the C. elegans Genome Sequencing Consortium. However, there was some discrepancy between the amino acid sequence of the product F31B12.1, predicted by the Consortium with the Genefinder program, and that deduced from our cDNA sequence. This was due to incorrect prediction of the exon-intron boundaries by the program.

Direct and GTP-dependent Association of PLC210 with Ras-- To map the Ras-binding site of PLC210, the insert of pACT9-1 was divided into two fragments, corresponding to residues 1112-1571 and residues 1570-1861 (Fig. 2A). Each region was tested for the ability to bind LET-60 in the two-hybrid assay. The result indicated that the Ras binding activity of PLC210 resided in residues 1570-1861 but not in residues 1112-1571 (Fig. 2A). Next, residues 1570-1861 were expressed as an MBP fusion in E. coli and tested for direct association with Ha-Ras in vitro (Fig. 2B). MBP-PLC210(1570-1861) bound preferentially to the GTPgamma S-loaded Ha-Ras. A clue for further narrowing down the Ras-binding domain of PLC210 came from a computer algorithm-based study by Ponting and Benjamin (23). They proposed the existence of a motif of roughly 100 amino acid residues, called the RA domain, which was conserved among Ras-binding regions of mammalian RalGDS and AF-6, and identified a variety of other proteins bearing this domain by searches of data bases. F31B12.1 was among them and was predicted to contain a tandem array of two complete RA domains. However, our cDNA sequence indicated that PLC210 actually lacked the C-terminal 14 residues of the downstream RA domain, rendering it incomplete. The fragment corresponding to residues 1570-1861 was further divided by XbaI digestion into two subfragments containing the individual RA domain (RA1 or RA2) (Fig. 1A and 2A), and the subfragments were examined for in vitro association with Ha-Ras (Fig. 2B). MBP-PLC210(1570-1670), containing RA1, was capable of binding Ha-Ras in a GTP-dependent manner with an affinity comparable with that of MBP-PLC210(1570-1861). However, MBP-PLC210(1669-1861) did not bind Ha-Ras at all. These results indicate that RA1 is responsible for Ras binding. This is the first example of a predicted RA domain that is shown experimentally to act as a functional Ras-binding domain.


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Fig. 2.   Direct interaction of PLC210 with Ras proteins. A, shown are the structures of PLC210 C terminus and regions covered by pACT4-2, pACT9-1, and their deletion derivatives. Numbers besides the bars represent the corresponding amino acid residues. Restriction cleavage sites are indicated. The interactions of these regions with LET-60 were examined by the yeast two-hybrid assay: +, positive interaction; -, no interaction. B, 10 pmol of the GTPgamma S-bound Ha-Ras (T) or the GDP-bound Ha-Ras (D) were incubated with 100 pmol of various MBP-PLC210 fusion proteins immobilized on amylose resin. The numbers on the top indicate the range of the expressed PLC210 in amino acid positions. Ha-Ras bound to each MBP fusion protein (upper panel) and a 1:10 aliquot of Ha-Ras used in each binding assay (lower panel) were detected by Western immunoblotting with the anti-Ras monoclonal antibody F235. Similar results were obtained in three independent experiments. C, adenylyl cyclase activity dependent on the GTPgamma S-bound Ha-Ras (80 nM) was subjected to inhibition by 500 nM each of MBP or MBP-PLC210(1570-1861). One unit of activity is defined as 1 pmol of cAMP formed in 1 min of incubation with 1 mg of proteins at 30 °C under standard assay conditions. Assays were repeated three times giving essentially similar results.

Role of Ras Effector Region in Association with PLC210-- To provide further support that PLC210 is a Ras effector, we examined the role of the Ras effector region in association with PLC210 by the following two experiments. First, we tested whether mutations within the effector region of Ha-Ras affect the association with PLC210. As shown in Table I, Ha-Ras mutants Y32F and T35S failed to bind to PLC210. The same Ha-Ras mutants failed to bind to Byr2, a Ras effector in Schizosaccharomyces pombe in the two-hybrid assay (18, 24). On the other hand, Ha-Ras mutants E37G and D38N, which failed to bind to Raf-1 (18, 25), did bind to PLC210. E37G was reported to bind to RalGDS and AF-6 carrying the RA domains (26, 27). Next, we tested whether PLC210 competes with S. cerevisiae adenylyl cyclase for binding Ras. The yeast adenylyl cyclase is a Ras effector whose mode of interaction with Ras had been characterized extensively; the GTP-bound Ras interacts with the leucine-rich repeat domain of adenylyl cyclase through its effector region and directly activates it in vitro (28). Further, we had shown that Raf-1, B-Raf, and Byr2 were capable of inhibiting this Ras-dependent activation of adenylyl cyclase by competitive sequestration of Ras (29-31). Accordingly, we tested the effect of added MBP-PLC210(1570-1861) on the Ras-dependent activation of adenylyl cyclase (Fig. 2C). The addition of MBP-PLC210(1570-1861) markedly inhibited the reaction, whereas that of MBP alone did not. The results of the two experiments indicate that PLC210 indeed interacts with the effector region of Ras.

                              
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Table I
Interactions of Ha-Ras effector region mutants with PLC210 and other effectors
The interactions were examined by the yeast two-hybrid assay. +, positive interaction; -, no interaction. The data on Raf-1, Byr2, RalGDS, and AF-6 are taken from Refs. 18, 25, 26, and 27.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Activation of PI-PLC is one of the early cellular responses to various extracellular signals. The activated PLC catalyzes the hydrolysis of PIP2 to generate the second messengers IP3 and diacylglycerol. IP3 induces the release of Ca2+ from intracellular stores, whereas diacylglycerol activates protein kinase C. Increases in cytoplasmic Ca2+ concentration and activation of protein kinase C have been implicated in diverse cellular events such as proliferation, differentiation, secretion, and migration (for a review see Ref. 32). By using the yeast two-hybrid screening, we have identified a C. elegans PI-PLC as a LET-60 Ras-binding protein. This PI-PLC, PLC210, bound mammalian Ha-Ras and a Ras homolog Rap1A as well but did not bind other small guanine nucleotide-binding proteins such as RalA, RhoA, Rac1, and Cdc42 by yeast two-hybrid analysis.2 The observations that PLC210 directly associates with Ras in a GTP-dependent manner, that this association requires the intact effector region of Ras, and that PLC210 competes with yeast adenylyl cyclase for binding Ras strongly suggest that PLC210 is a novel Ras effector. Presently, we do not know how Ras regulates PLC210. A preliminary experiment where the GTP-bound Ras was added in vitro to MBP-PLC210(612-1861) failed to observe any effect on the PLC activity.2 It could be that Ras can stimulate enzymatic activity of PLC210 only in cooperation with some unknown factor that was lacking in our assay. Alternatively, association with Ras might simply translocate PLC210 to a specific membrane compartment containing appropriate substrates.

It is unclear whether a mammalian homolog of PLC210 exists. In mammalian cells, an increase in PI-PLC activity upon treatment with growth factors has been largely attributed to activation of PLC-gamma , an isoform of PI-PLC; PLC-gamma interacts directly with the activated receptors tyrosine kinases and is activated by phosphorylation (32). However, the observation that the rate of phosphoinositide turnover in Ras-transformed cells was three times that in untransformed cells implied a persistent stimulation of PI-PLC in these cells (33). Further, injection of anti-PI-PLC antibody has been shown to inhibit Ras-induced mitogenesis (34). These observations are consistent with the possibility that an as yet unidentified species of PI-PLC regulated by Ras plays some role in mammalian cell proliferation, although they might be explained by accessory events accompanying the Ras-induced transformation such as autocrine stimulation of receptors coupled to known PI-PLC.

In this regard, it may be interesting to note that PLC210 interacts with Ha-Ras E37G but not with T35S. Although these mutants by themselves possess poor transforming activities on NIH3T3 fibroblasts, they exhibit a strong transforming activity when expressed together (25-27). This phenomenon has been accounted for by cooperation of the two distinct Ras effectors, Raf-1 and RalGDS, because Ha-Ras T35S can interact with Raf-1 but not with RalGDS and E37G can interact with RalGDS but not with Raf-1 (26, 27). However, it could also be explained by a cooperation between Raf-1 and another Ras effector with a Ras binding property similar to that of RalGDS. In this sense, the mammalian homolog of PLC210, if present, could be a reasonable candidate to test its cooperation with Raf-1 in cellular transformation. In fact, a number of reports indicate that constitutive activation of PI-PLC leads to growth factor-dependent and -independent cellular transformation in NIH3T3 cells (32).

All known PI-PLCs can be divided into three classes (beta , gamma , and delta ) exemplified by the 150-kDa PLC-beta 1, the 145-kDa PLC-gamma 1, and the 85-kDa PLC-delta 1 on the basis of size, immunological reactivity, and amino acid sequence (35). PLC210 differs from members of these classes not only in its size but also in its overall structure: (i) The region N-terminal to the X, Y, and C2 domains of PLC210 (~900 residues) is longer than those of the other PI-PLCs (~300 residues) and harbors the unique CDC25-like domain. (ii) PLC210 has ~200 residues linking the X and Y domains, which are different in size from those of PLC-beta and PLC-delta isoforms (40-110 residues) and of PLC-gamma isoforms (~400 residues). This region does not contain any known structural motif such as an array of SH2/SH3 domain found in PLC-gamma isoforms. (iii) In comparison with the PLC-gamma and PLC-delta isoforms, the PLC-beta isoforms contain an additional regulatory C-terminal region (200-300 residues) that is responsible for the specific binding to and activation by the alpha  subunit of Gq. Although the C-terminal region of PLC210 is comparable in length, it has a distinct structure for Ras binding, the RA domain. These observations strongly suggest that PLC210 represents a prototype of a novel class of PI-PLC.

    ACKNOWLEDGEMENTS

We thank Dr. R. Barstead for providing lambda ACT-RB2 library, Dr. Y. Kohara for providing C. elegans cDNA library, Dr. T. Takenawa for providing pGEX-PLC-delta 1, and Dr. H. Sakamoto (Kobe University) for useful instruction in the yeast two-hybrid system. We also thank X.-H. Deng for skillful technical assistance and A. Seki and Y. Kawabe for help in preparation of this manuscript.

    FOOTNOTES

* This work was supported by grants-in-aid for scientific research in priority areas and for scientific research (B) and (C) from the Ministry of Education, Science, and Culture of Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Diseases and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF044576.

Dagger To whom correspondence should be addressed. Tel.: 81-78-341-7451, Ext. 3230; Fax: 81-78-341-3837; E-mail: kataoka{at}kobe-u.ac.jp.

1 The 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; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); RA, Ras-associating.

2 M. Shibatohge, K. Kariya, and T. Kataoka, unpublished observation.

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Abstract
Introduction
Procedures
Results
Discussion
References

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