From the Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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Two-hybrid Screening--
ACT-RB2, a random-primed mixed
stage C. elegans cDNA library constructed in
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
-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-1 (provided by Dr. Tadaomi Takenawa, University of Tokyo,
Japan) was used for expression of the full-length rat PLC-
1 as a GST
fusion in E. coli. MBP-PLC210(612-1861) and GST-PLC-
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 -galactosidase activity was
measured by blue color development after incubation with
5-bromo-4-chloro-3-indolyl
-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 GTPS-bound Ha-Ras and of its inhibition by
the purified MBP-PLC210(1570-1861) were carried out as described
previously (20).
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RESULTS |
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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-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-
1, 9.1 nmol/min/mg protein, obtained in the same assay condition.
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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 GTPS-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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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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DISCUSSION |
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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-, an
isoform of PI-PLC; PLC-
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 (,
, and
)
exemplified by the 150-kDa PLC-
1, the 145-kDa PLC-
1, and the
85-kDa PLC-
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-
and PLC-
isoforms (40-110 residues) and of PLC-
isoforms (~400
residues). This region does not contain any known structural motif such
as an array of SH2/SH3 domain found in PLC-
isoforms. (iii) In
comparison with the PLC-
and PLC-
isoforms, the PLC-
isoforms
contain an additional regulatory C-terminal region (200-300 residues)
that is responsible for the specific binding to and activation by the
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.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Barstead for providing
ACT-RB2 library, Dr. Y. Kohara for providing C. elegans
cDNA library, Dr. T. Takenawa for providing pGEX-PLC-
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.
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FOOTNOTES |
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* 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.
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; GTPS, guanosine 5'-O-(3-thiotriphosphate); RA, Ras-associating.
2 M. Shibatohge, K. Kariya, and T. Kataoka, unpublished observation.
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REFERENCES |
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