(Received for publication, June 28, 1995; and in revised form, July 31, 1995)
From the
RalA and RalB are GTPases of unknown function and are activated by proteins, RalGDS, that interact with the active form of another GTPase, Ras. To elucidate Ral function, we have searched for proteins interacting with an activated form of RalA using the two-hybrid method and a Jurkat cell library. We have identified a partial cDNA encoding a protein, RLIP1, which binds to activated RalA and this binding requires an intact effector domain of RalA. Biochemical data with purified RalA confirm the genetic results. This protein also bears a region of homology with GTPase-activating protein (GAP) domains that are involved in the regulation of GTPases of the Rho family and, indeed, RLIP1 displays a GAP activity acting upon Rac1 and CDC42, but not RhoA. This GAP region is not required for RLIP1 binding to Ral.
The whole cDNA
was cloned, and it encodes a 76-kDa polypeptide, RLIP76, which also
binds RalA. The Rho pathway is involved in membrane and cytoskeleton
modifications after mitogenic stimulation and acts in parallel to and
synergistically with the Ras pathway. We propose that these pathways
are linked through a cascade composed of Ras RalGDS
Ral
RLIP76
CDC42/Rac1/Rho, allowing modulation of the Rho
pathway by the Ras pathway.
Ral proteins are biochemically well characterized GTPases whose
functions have long remained elusive(1, 2) . A
potential clue was provided by the finding that RalGDS ()and
a RalGDS-like protein, which are activators of RalA and
RalB(3) , interacts with the activated form of Ras and that
this interaction requires the integrity of the Ras effector
domain(4, 5, 6) . Thus RalGDS, and therefore
Ral proteins, might be involved in transducing pathways that signal
through Ras.
In order to decipher Ral function, we have searched for proteins that interact with the activated form of RalA. Using a two-hybrid method and a mutant of RalA deficient in its intrinsic GTPase activity (RalAV23), we have isolated a partial cDNA encoding a protein (RLIP1, Ral interacting protein 1) that has characteristics of a Ral effector protein.
The whole cDNA was isolated and sequenced; it contains an ORF encoding a predicted 76-kDa protein (RLIP76) that binds RalA.
Out of the Ral binding region, RLIP76 contains a GAP region related to RhoGAP domains and this structural homology reflects a functional homology with a GAP activity acting upon CDC42HS and Rac1.
Yeast and the two-hybrid procedures were handled according
to published methods(9, 10) . Library plasmids from
transformed yeast colonies were recovered using HB101 as a recipient
strain, selected on M9 medium lacking leucine. When mating was used for
two-hybrid tests, strain L40 was mated with strain AMR70 (MAT,
leu2, trp1, his3, ade2, URA3::lexAop-lacZ) (a gift from
S. Fields). When two-hybrid results are presented, we are showing the
results of
-galactosidase test on filter paper. There was no
discrepancy between the His auxotrophy test and the
-galactosidase
test.
When required, point mutations were introduced using the Transformer site-directed mutagenesis kit (Clontech). Any DNA fragment submitted to mutagenesis and all PCR products were sequenced.
Transcription and translation in presence of methionine were performed using 1 µg of PCR product, and, sequentially, a mRNA capping kit and an in vitro translation kit (Stratagene).
In vitro binding studies
after guanine nucleotide exchange were performed as described above
with the following alterations; 10 µg of glutathione-Sepharose
4B-bound proteins were washed twice in ice-cold exchange buffer (20
mM Tris-HCl, pH 7.5, 10 mM EDTA, 5 mM
MgCl, 1 mM dithiothreitol, 1 mM Pefabloc), and incubated for 3 h at 4 °C in 50 µl of
exchange buffer containing 90 µM GTP or GDP. The
nucleotide exchange reaction was stopped by adding MgCl
to
20 mM followed by two washes with binding buffer containing 20
mM MgCl
.
In all cases, protein concentrations were estimated by Coomassie Blue staining of SDS-PAGE gels and adjustments were made for the same amount to be used in all experiments.
Ral allele dependence of the interaction was checked using pRLIP1.
RalAV23, RalAV23A46 (an effector domain mutant), RalAA26 (which mimics
a Ras GDP-blocked mutant), and RalAV23A26 were cloned in pBTM116. Fig. 1A shows the signals displayed in a
-galactosidase test. First, RLIP1 is able to interact not only
with RalAV23
CT (data not shown) but also with RalAV23 and RalAwt.
Second, RLIP1 interaction with RalAA26 or with RalAV23A26 is
undetectable. Since a G26A mutation is supposed to block RalA in a
GDP-bound state, this result suggests that RLIP1 binds to RalA only
when this latter is bound to GTP and not to GDP. Third, RLIP1 is unable
to interact with a RalAA46 mutant. Based on sequence and structure
similarities with c-Ha-Ras, a T46A RalA mutant would have an impaired
effector domain. This result suggests that RalA requires an intact
effector domain to bind to RLIP1. It also suggests that, in yeast,
LexA-Ralwt is, at least in part, in the GTP-bound conformation, as is
the case for other GTPases expressed as LexA fusions(9) .
Figure 1:
Allele-specific interaction of RalA and
Rac1 with RLIP1. L40 yeast cells were transformed with pairwise
combinations of plasmids expressing proteins fused to LexA from a TRP1
plasmid and proteins fused to the activation domain of Gal4 (GALAD) from a LEU2 plasmid. Transformed cells were patched
on selective medium (DO-WL), then replicated on a Whatman No.
40 paper laid on a DO-WL plate. After 24 h, a -galactosidase
activity assay on paper was performed. In A and B,
alleles of RalA and of Rac1 were expressed as LexA fusion proteins,
respectively. Western blot analysis has shown that Ral alleles are
expressed at a similar level, as are Rac alleles (data not
shown).
From these data, it emerges that RLIP1 is a good candidate to be an ``effector'' of RalA function.
Figure 2:
RalA
and RLIP1 interact in vitro. A, in vitro synthesized S-RLIP1 was run on a SDS-PAGE (10%
acrylamide) gel. Autoradiography shows that RLIP1 migrates as a 66-kDa
protein. B and C, equal amounts of
S-RLIP were incubated with glutathione-Sepharose beads
bound to equal amounts of GST, GST-RalA, or GST-RalAV23, preloaded (C) or not (B) with GDP or GTP (see
``Experimental Procedures''). After overnight binding at 4
°C, beads were centrifuged and the supernatant removed. Beads were
washed three times and boiled in SDS sample buffer. Half of the beads
(the bound fraction, b) and half of the supernatant (the
unbound fraction, u) were analyzed by SDS-PAGE followed by
autoradiography.
When in vitro guanine nucleotide exchange was performed prior to RLIP1 binding, RLIP1 again did not bind to GST. It did not bind GST-RalA or GST-RalAV23 loaded with GDP. It bound GST-RalA and, even better, GST-RalAV23 loaded with GTP (Fig. 2C). These data show that RLIP1-RalA interaction is not mediated by a yeast protein and are consistent with the genetic results, i.e. RLIP1 interacts directly with the GTP-bound form of RalA whose effector domain is required.
In the Ras superfamily, within the Ras branch (to which RalA belongs; (18) ), RLIP1 was not able to interact with c-Ha-Ras or with Rap1A, Rap2A, or Rap2B. However, it does interact with RalB.
In the Rab branch, RLIP1 was not able to interact with Rab5, Rab6, Rab7, or Rab13.
Finally, in the Rho/Rac branch, we were not able to detect any interaction with RhoA, RhoB or RhoG, but RLIP1 is able to interact with Rac1 (Fig. 1B).
This latter interaction was further investigated using Rac1 alleles. Fig. 1B shows that RLIP1 is able to interact with Rac1V12S189 but not with Rac1V12N17S189, a dominant negative mutant blocked in the GDP-bound form, or Rac1V12A35S189 and Rac1V12A38S189, two effector domain mutants(19) . These results suggest that Rac1 bound to GTP is able to interact with RLIP1 through its effector domain.
Figure 3:
GTPase activating activity of RLIP1 upon
Rac1 and CDC42. Purified Rac1, CDC42, and Rap2A proteins were loaded
with [-
P]GTP, and GTPase activity was
assessed by a filter binding assay(30) . 100% refers to the
radioactivity bound to the protein at time 0. For each protein, the firstcolumn reflects the intrinsic GTPase activity,
the second the GTPase activity in presence of RLIP1. For CDC42
and Rac1, the thirdcolumn reflects the GTPase
activity in presence of the GAP region of Bcr, which harbors a powerful
GAP activity upon Rac1 and CDC42.
Two consecutive
rounds of 5`-RACE were required to obtain the full-length cDNA that was
also recovered from a skeletal muscle cDNA library in gt10 and
from a placenta cDNA library in
EXlox. The sequence of the
full-length cDNA was established.
There is one main reading frame (ORF), from base 224 to base 2188, preceded by a correct translation initiation sequence(21) . Two short ORFs (13 and 3 codons) are found within the 5` end of this cDNA but none of them is preceded by a correct translation initiation sequence. A 1664-base pair non-coding sequence is found 3` to the ORF.
This ORF encodes a protein made of 655 amino acids and of predicted molecular mass 76 kDa that we named RLIP76 (Fig. 4A). RLIP1 in plasmid pRLIP1 starts at amino acid 185, and RLIP2 starts at amino acid 403.
Figure 4:
Sequence and analysis of RLIP76. A, amino acid sequence of RLIP76. B, homology between
RLIP76 and GAP domains Both a Blitz homology search program (EMBL) and
a BLAST homology search program (NCBI) aligned a region of RLIP76 with
domains of proteins that display a CDC42/Rac/Rho GAP activity. The two
best scores were obtained with Bcr-GAP region and n-chimaerin,
and these alignments are shown here. Identical residues are bold, conserved residues are indicated by a + on the
consensus line. Blocks 1, 2, and 3 refer to
three blocks conserved among proteins of this class(22) . C, deletion analysis. RLIP76 coding region was inserted in
plasmid pGAD1318, which allows expression of GAL4AD fusion proteins.
From pRLIP1 isolated during the screen, different regions were deleted.
For each construct, the junction region was sequenced. These plasmids
were tested for their ability to elicit a positive signal in the
two-hybrid system in presence of a plasmid expressing a LexA-RalA
protein or, when indicated, a LexA-Rac1 protein. D, functional
regions. Based on the previous analysis (B and C),
two regions can be defined functionally: a GAP region from residue 210
to 353, and a RalA binding region from residue 403 to 499. Compilation
of results from three different programs predicting secondary
structures (31, 32, 33) led to this scheme.
The two regions represented by boxes are predicted by at least
two of the programs to be composed mainly of -helices. In
addition, part of the second region is predicted to be superfolded as a
coiled-coil structure(32) .
Data bank comparison revealed that the region extending from amino acid 210 to amino acid 353 shares significant homology to regions of proteins bearing a CDC42/Rho/Rac-GAP activity, like Bcr, chimaerins, Drosophila rotund, and the Ras-GAP-binding protein p190(22) . Fig. 4B shows this striking homology with Bcr and n-chimaerin.
Fig. 4C gives the
results obtained with two-hybrid plasmids expressing different parts of
RLIP76. These results allow definition of the maximum size of the
region required for RalA binding. Together with secondary structure
predictions (Fig. 4D), the overall structure of RLIP76
can be depicted schematically as composed of four regions: an
N-terminal region where amino acids 65-170 are predicted to be
structured in -helices, the GAP-like region (aa 210-353),
the Ral binding region (aa 403-499) predicted to be composed in
part of
-helices, and a C-terminal region (aa 499-655). Part
of this latter region and part of the Ral binding region (aa
440-610) are predicted to be able to form a coiled-coil
structure.
By FISH analysis of 19 R-banded metaphase cells, RLIP1 gene was localized on band 18p11 (25 chromosomes positive on both chromatid out of 38). A minor localization on band 3q26 was also detected (9 out of 38) that might suggest the existence of a related gene (data not shown).
We have identified a cDNA encoding a protein, RLIP1, that is able to interact with RalA and RalB, and which has the characteristics of a Ral effector; biochemical data and genetics suggest that RLIP1 binds better to Ral-GTP than to Ral-GDP and that this interaction requires a functional effector domain. According to Northern blot analysis, RLIP1 is ubiquitously (but at low levels) expressed, as are Ral and RalGDS, a Ral activator(3) .
Although able to discriminate Ral from other GTPases, RLIP1 also binds to the active form of Rac1. We suppose that the molecular avatar of this binding is a GAP-like region whose absence impairs interaction with Rac1 but not with RalA; domains involved in Rac binding and in Ral binding are physically distinct. We also show that the structural homology with GAP-like regions reflects a functional homology. RLIP1 is able to activate specifically hydrolysis of GTP bound to Rac1 and to CDC42, but not, as expected, to Rap2A.
The whole cDNA was cloned; it encodes a 76-kDa protein, RLIP76, able to bind to RalA as based on a two-hybrid assay.
These findings raise several questions. Our results allow us to conclude that the GAP-like region of RLIP76 displays a bona fide GAP activity acting upon CDC42 and Rac1. However, this GAP activity is rather weak when compared to the GAP activity of Bcr tested in parallel. This could be due either to technical problems (we do not know how much of purified RLIP is active), to structural problems (either a larger part or a smaller part of RLIP76 could do better) or to biological constraints (a companion protein might increase this activity). These considerations lead to more questions. What is Ral doing to RLIP76? Is it localizing RLIP76 in the vicinity of its target, as happens to be the case for other GTPases involved in the subcellular localization of certain effectors(23, 24) , and/or is it modulating RLIP76-GAP activity?
Ras and Rho pathways are both activated during mitogenic signaling through transmembrane receptors. It is unclear if their activation is sequential or parallel, but they seem to work synergistically(19, 25, 26, 27, 28, 29) . The Rho pathway, a cascade of GTPases, from CDC42 to Rho passing by Rac, acts upon structures involved in cell shape plasticity. Activation of Ras leads to several cytoplasmic and nuclear phenomena as well as membrane modifications. And Ral proteins are potentially switched to their active form through interaction of activated Ras with Ral activators. We propose that RLIP76 participates in the cross-talk between these GTPase cascades, modulating the state of activity of the CDC42/Rac/Rho pathway in response to Ras activation.
Finally,
regions of RLIP76 seem a priori not to participate in the
above functions. The -helix-rich regions, especially the
coiled-coil region, might be involved in interactions with other
proteins. Alternatively, the coiled-coil region might participate in
the homodimerization of RLIP76. After Ras and subsequent Ral
activation, Ral binding to RLIP76 could separate the monomers and
render the GAP catalytic region accessible to its target.
It will be of great interest in future RLIP studies to analyze the regulation and interplay of the various separate functional domains.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42542[GenBank].