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Bimodal regulation of RAF by CNK in Drosophila

2003; Springer Nature; Volume: 22; Issue: 19 Linguagem: Inglês

10.1093/emboj/cdg506

1460-2075

Mélanie Douziech,

Ubiquitin and proteasome pathways

Article1 October 2003free access Bimodal regulation of RAF by CNK in Drosophila Mélanie Douziech Mélanie Douziech Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author François Roy François Roy Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Gino Laberge Gino Laberge Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Martin Lefrançois Martin Lefrançois Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Anne-Valérie Armengod Anne-Valérie Armengod Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Marc Therrien Corresponding Author Marc Therrien Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Mélanie Douziech Mélanie Douziech Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author François Roy François Roy Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Gino Laberge Gino Laberge Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Martin Lefrançois Martin Lefrançois Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Anne-Valérie Armengod Anne-Valérie Armengod Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Marc Therrien Corresponding Author Marc Therrien Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada Search for more papers by this author Author Information Mélanie Douziech1, François Roy1, Gino Laberge1, Martin Lefrançois1, Anne-Valérie Armengod1 and Marc Therrien 1 1Clinical Research Institute of Montreal, Laboratory of Intracellular Signaling, 110 Pine Avenue, West Montreal, PQ, H2W 1R7 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5068-5078https://doi.org/10.1093/emboj/cdg506 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in Drosophila. Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, we demonstrate that CNK has antagonistic properties with respect to RAF activity. We show that CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, we also report that the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module. Introduction The extracellular-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) module, herein referred to as the MAPK module, is defined as a group of three kinases that is comprised of specific isoforms of the serine/threonine kinase RAF, the dual-specificity MAPK/ERK kinase (MEK) and the proline-directed serine/threonine kinase ERK/MAPK. This module transmits signals mainly received from the small GTPase RAS to control a number of critical cellular events such as proliferation, differentiation and survival (for review, see English et al., 1999). Early characterization of this signaling pathway identified a simple relationship among the core components, whereby upon RAS activation, RAF is recruited to the plasma membrane by RAS, which in turn triggers a phosphorylation cascade from RAF to MAPK. In-depth investigations of each individual step, however, are now unraveling a surprisingly complex process that involves additional proteins whose respective role is either partially or not understood (for review, see Kolch, 2000). RAS was recognized early on as a major player in RAF activation, principally by its ability to recruit RAF to the plasma membrane through an interaction between its effector loop region and the RAS-binding domain (RBD) on RAF (for review, see Avruch et al., 1994). However, as this event did not appear sufficient to activate mammalian Raf-1 in vitro, additional molecules were predicted to participate in RAF activation. A search for proteins that could bind and modify RAF activity identified the 14-3-3 protein family as potential RAF regulators (for review, see Morrison and Cutler, 1997). These abundant proteins bind as dimers a wide range of targets through the recognition of specific sequence motifs, some of which require threonine or serine phosphorylation for binding (for review, see Tzivion and Avruch, 2002). RAF proteins contain two evolutionarily-conserved 14-3-3 binding sites; one surrounding phospho-serine 259 (pS259) and the second at phospho-serine 621 (pS621) in Raf-1 (Muslin et al., 1996). Growing evidence now suggests that 14-3-3-binding to these sites has opposite effects on RAF. Whereas pS621 occupancy seems critical for RAF activity (Thorson et al., 1998; Yip-Schneider et al., 2000), pS259 binding correlates with inactive RAF (Dhillon et al., 2002; Light et al., 2002), possibly by forcing RAF to adopt an inactive conformation and/or by sequestering RAF in the cytoplasm. Displacement of 14-3-3 from pS259, an event apparently triggered by RAS-binding and accompanied by pS259 dephosphorylation, appears to be one of the critical events leading to RAF activation. Despite its importance, this event does not fully account for RAF activation since mutations disrupting the pS259 site modestly enhance RAF catalytic function (Dhillon et al., 2002; Light et al., 2002). Genetic and yeast two-hybrid screens conducted over the years have identified additional putative components of the RAS/MAPK pathway (for review, see Kolch 2000). As some of these appear to modulate RAF function, their molecular characterization might unveil key aspects to solve at last the mystery surrounding RAF activation. For instance, genetic screens in Drosophila and Caenorhabditis elegans identified kinase suppressor of Ras (ksr), an evolutionarily conserved gene encoding a putative protein kinase structurally related to RAF (Kornfeld et al., 1995; Sundaram and Han, 1995; Therrien et al., 1995). Functional studies revealed that KSR facilitates signaling from RAF to MAPK essentially by its ability to bring together the three kinases of the MAPK module (for reviews, see Morrison, 2001; Raabe and Rapp, 2002; Roy and Therrien, 2002). Besides its importance for efficient MEK and MAPK activation, KSR also appears to control RAF activity since depletion of endogenous KSR by RNA interference (RNAi) impaired RAF catalytic function in Drosophila S2 cells (Anselmo et al., 2002). It is unclear, however, whether this effect depends on KSR's scaffolding property as recently suggested (Roy et al., 2002) or is mediated by another mechanism. Another potential RAF regulator is connector enhancer of KSR (CNK), a multidomain-containing protein conserved among metazoans, which was originally identified in a KSR-dependent genetic screen in Drosophila (Therrien et al., 1998). As for other bona fide components of the RTK/RAS/MAPK pathway in Drosophila, CNK is required for photoreceptor cell differentiation, wing vein formation as well as for imaginal disc cell proliferation and/or survival (Therrien et al., 1998). Genetic epistasis experiments positioned CNK downstream of RAS, but upstream or in parallel to RAF, thereby suggesting that CNK might be regulating RAF activity (Therrien et al., 1998). Consistent with that possibility, CNK was found to associate with the catalytic domain of RAF (Therrien et al., 1998) and depletion of endogenous CNK by RNAi in S2 cells abolished insulin-induced RAF activation (Anselmo et al., 2002). The role of CNK with respect to RAF is probably not restricted to Drosophila since a rat homolog, named Maguin, has recently been found to associate with Raf-1 in rat brain extracts (Yao et al., 2000). Here, using a CNK-dependent MAPK activation assay in S2 cells combined to a novel RNAi-based rescue protocol, we show that CNK has both a positive and a negative impact on RAF function. We found that CNK, through two of its N-terminal domains, integrates RAS signals to control MEK phosphorylation by RAF. In contrast, we found that CNK's ability to associate with RAF is mediated by a short bipartite element that acts as an inhibitor of RAF catalytic function. Finally, we present evidence that the opposite functions of CNK amplify signaling difference between the off and on states of a KSR/RAF/MEK complex, which might contribute to the switch-like behavior of the MAPK module. Together, these findings identify CNK as a novel type of signal regulator that specifically controls RAF function. Results To delineate biochemically the position of CNK with respect to the components of the RAS/MAPK module, we depleted endogenous CNK by RNAi in a stable RASV12-expressing S2 cell line and assessed its effect on endogenous MEK and MAPK activation. As shown in Figure 1A, reduction of CNK by the addition of double-stranded (ds) CNK RNA specifically abrogated MEK and MAPK activation, as revealed by the decrease in phosphorylated (activated) MEK and MAPK. These results demonstrated that CNK is required downstream of RAS for activation of the MAPK module. We next examined the effect of removing CNK on activated RAF-induced MAPK activation. Compared to the activated receptor tyrosine kinase Sevenless (SevS11) or RASV12, which did not activate MAPK upon CNK or MEK depletion (Figure 1B, lanes 3, 4, 6 and 7), activated RAF (Tor4021RAFc) was still fully capable of activating MAPK upon CNK depletion, but not when MEK was eliminated (Figure 1B, lanes 9 and 10). Together, these results strongly suggest that CNK is acting between RAS and RAF. Figure 1.CNK activity is required downstream of RAS, but upstream of RAF. (A) Untreated (−) or CuSO4-treated (+) RASV12 cells were either incubated in the absence (−) or in the presence (+) of the indicated dsRNAs. pMEK and pMAPK levels, as well as endogenous RAS, RAF, MEK, MAPK and CNK levels were assessed by immunoblot analysis using the antibodies indicated to the right. The results shown here and thereafter are representative of at least three similar experiments. (B) S2 cells were transfected with the haMAPK reporter construct (0.3 μg) either alone (lane 1) or together (+) with the indicated combinations of SEVS11 (0.4 μg), haRASV12 (0.4 μg) or Tor4021RAFc (0.2 μg) constructs and the dsCNK or dsMEK RNAs (0.5 μg). Cells were lysed 16 h post-induction of expression and pMAPK levels were determined. Protein levels were determined as indicated. (C) Three milligrams of protein from plain S2 cells or 0–14 h Drosophila embryos were immunoprecipitated (IP) using either α-RAF or α-CNK antibodies. Download figure Download PowerPoint Overexpression of CNK has been found previously to associate with endogenous RAF in S2 cells. Furthermore, a C-terminal fragment of CNK has also been reported to interact directly with the catalytic domain of RAF (Therrien et al., 1998). To demonstrate that a CNK/RAF complex does exist in vivo, we immunoprecipitated plain S2 cell or Drosophila embryo extracts using either anti-RAF or anti-CNK antibodies and probed immunoblots with either antibodies to look for co-immunoprecipitation. As shown in Figure 1C, the anti-RAF antibodies brought down endogenous CNK (∼10% of total NP-40-soluble CNK) and, likewise, the anti-CNK monoclonal antibody co-immunoprecipitated endogenous RAF (∼5% of total NP-40-soluble RAF) in both S2 cells and embryos. These results thus demonstrate the existence of a CNK/RAF complex in vivo. Together with the fact that CNK activity appears to be required upstream of RAF, these findings strongly suggest that CNK directly regulates RAF function. CNK has opposite effects on RAF function To characterize the molecular event(s) within the RAS/MAPK pathway that is/are regulated by CNK activity, we examined whether forced expression of CNK could modulate MAPK activation in S2 cells. For this purpose, we used three Flag epitope-tagged CNK constructs (Figure 2A), namely, full-length (FL), N-terminal (NT) and C-terminal (CT) that had previously been shown to modulate RASV12-mediated signaling in the developing Drosophila eye (Therrien et al., 1999). Figure 2.Opposite behavior of CNK in the RAS/MAPK pathway. (A) Schematic representation of Drosophila FL–CNK (top open box) with its various domains/elements (black boxes): sterile alpha motif (SAM); conserved region in CNK (CRIC); PSD-95/DLG-1/ZO-1 (PDZ); proline-rich stretch (Pro); pleckstrin homology (PH); RIR; RIM; IS. Numbers on top correspond to amino acid positions frequently referred to in the text. Solid lines, labeled to the left, denote the various CNK deletion constructs used in this study. Every CNK construct contains one copy of the Flag epitope at the N-terminus. (B) S2 cells were transfected with haMAPK alone (lane 1) or with the indicated combinations of haRASV12 (0.25 μg), FL–CNK (FL; 1.65 μg), NT–CNK (NT; 0.1 μg) and CT–CNK (CT; 1.65 μg) constructs. Cells were lysed 36 h post-induction and pMAPK levels were determined. Owing to a poor detection of Flag-tagged CNK constructs directly from cell lysates using the anti-Flag antibody, their levels were examined by immunoprecipitation (IP). (C) S2 cells were transfected with haMAPK alone (0.3 μg) or with the indicated combinations of Tor4021RAFc (0.2 μg), mycMEK2E (0.3 μg), FL (0.6 μg) or NT (0.1 μg) constructs. Cells were lysed 16 h post-induction. The flag-tagged CNK variants were examined using anti-CNK. (D) S2 cells were transfected and analyzed as in (C) using the indicated combinations of haMAPK (0.6 μg), RASV12 (0.6 μg), RASV12S35 (0.1 μg), RASV12G37 (0.25 μg), RASV12C40 (0.6 μg) and NT (0.25 μg). Various amounts of the RAS constructs were used to adjust for their apparent difference in expression levels. The RAS proteins were not tagged and thus were monitored using an anti-Drosophila RAS monoclonal antibody, which also detected endogenous RAS as seen in lanes 1 and 2. (E) S2 cells were transfected using the indicated combinations of haMAPK (0.6 μg), haRASV12 (0.25 μg), and wild-type or mutant NT constructs (0.25 μg). The mutated SAM domain (SAMmut) has a two amino acid change in conserved residues (amino acids W17S and I18S) that are critical for structural integrity (Stapleton et al., 1999). For unclear reasons, this NT–CNK mutant migrates differently from the other NT constructs (lane 4). We have generated another mutant version of the SAM domain (L71K), which changes an amino acid shown to be critical for dimer formation of the EphA4 receptor SAM domain, but does not appear to alter the structural integrity of the domain (Stapleton et al., 1999). This mutated SAM domain (NTL71K–CNK) migrates normally, and like the SAMW17S–I18S mutant, it does not cooperate with RASV12 (data not shown). NTCRICmut has a three amino acid deletion (A162-H163-R164) in the CRIC region similar to the mutation found in a Drosophila cnk loss-of-function allele (Therrien et al., 1998). Finally, NTPDZmut has a two amino acid change (G217S and F218S) in highly conserved residues of the PDZ domains (Ponting et al., 1997). Download figure Download PowerPoint We assayed for MAPK activation by monitoring the phosphorylated levels of HA-tagged MAPK as performed above for endogenous MAPK. When expressed alone, none of the CNK constructs elevated phospho-MAPK (pMAPK) levels (data not shown and Figure 2D, lane 2 for NT–CNK). However, compared to HA-tagged RASV12 expressed alone (Figure 2B, lane 2), co-expression of FL–CNK and CT–CNK inhibited MAPK activation (Figure 2B, lanes 3 and 5, respectively), whereas NT–CNK stimulated MAPK activation (Figure 2B, lane 4). Therefore, these results indicate that forced expression of CNK affects RAS-mediated MAPK activation and also suggest that CNK comprises both positively- and negatively-acting regions. Because CNK appears to be required between RAS and RAF (Figure 1B), we reasoned that the opposite effects of CNK could be due to a modulation of RAF function. To investigate this possibility, we examined the ability of FL–CNK and NT–CNK to alter MAPK activation induced by activated RAF or activated MEK (myc-tagged MEK2E). If FL–CNK blocked a positive step upstream of RAF, there should be no effect on MAPK activation induced by activated forms of RAF or MEK. In contrast, if CNK blocked a step downstream of RAF, it should either inhibit RAF or both RAF and MEK activities depending on the position of the inhibitory event. Strikingly, we found that FL–CNK (like CT–CNK, data not shown), completely prevented MAPK activation induced by activated RAF (Figure 2C, lane 3), but not by activated MEK (lane 6) [the apparent slight positive effect of FL–CNK on pMAPK levels induced by MEK2E (lane 6) was not reproducible]. These results therefore indicate that the negative influence of CNK occurs at a step between RAF and MEK. We applied the same logic to position the positive effect of NT–CNK and concluded that NT–CNK exerts its positive effect in a RAS-dependent manner between RAS and RAF as NT–CNK was inert on its own and did not cooperate with either activated RAF or MEK (Figure 2C, lanes 4 and 7, respectively). Therrien et al. (1999) previously reported that NT–CNK cooperated in the Drosophila eye not only with RASV12, but also with RASV12G37, which is a RAS effector loop mutant that has a much reduced capacity to send signals through the MAPK pathway owing to its impaired association with RAF (White et al. 1995). They concluded that either NT–CNK augments RAS signaling through a RASV12G37-dependent, but MAPK-independent pathway or that, if NT–CNK functions between RAS and RAF, it could rescue or compensate to some extent the defect caused by this particular effector loop mutation thereby permitting RAF activation. To distinguish between these possibilities, we co-expressed NT–CNK with RASV12 or the three RAS effector loop mutations that had been tested. These included RASV12S35, which interacts normally with RAF, and RASV12G37 and RASV12C40, which no longer interact with RAF (data not shown). As shown in Figure 2D, NT–CNK strongly augmented pMAPK levels induced by either RASV12 or RASV12S35 (lanes 3–6) and surprisingly, it also allowed RASV12G37, which is inert on its own, to activate MAPK (compare lanes 7 and 8). These data therefore suggest that NT–CNK exerts its effect not through an alternate pathway, but largely within the RAS/MAPK pathway. The fact that NT–CNK appears to compensate to some extent the inability of RASV12G37 to activate MAPK, but not for RASV12C40 (Figure 2D, compare lanes 9 and 10), indicates that these two effector loop mutations are not equivalent with respect to their defect in activating RAF. NT–CNK comprises three conserved regions (SAM, CRIC and PDZ domains; Figure 2A). To determine which of these is required for the positive effect of NT–CNK on the MAPK module, we tested the activity of NT–CNK mutant constructs affecting each domain individually. When co-expressed with RASV12, the mutated SAM and CRIC domain variants failed to cooperate with RAS (Figure 2E, lanes 4 and 5). In contrast, the PDZ domain mutant still retained activity (Figure 2E, lane 6). These results thus indicated that the SAM and CRIC domains are critical for the ability of NT–CNK to stimulate MAPK activation by RAS. Since the CRIC mutation used in this assay corresponds to the lesion found in a cnk loss-of-function allele (Therrien et al., 1998), it strongly suggests that our assay mimics a genuine functional property of CNK (see below). Two short amino acid sequences in CNK define a ‘RAF-inhibitory region’ that blocks MEK phosphorylation by RAF Interestingly, in addition to its positive role on the MAPK module, CNK can block RAS- or RAF-dependent MAPK activation (Figure 2). We investigated this property by first mapping the region(s) of CNK that has a negative influence on the pathway. We generated a series of C-terminal deletions of CNK (Figure 2A) and tested their ability to inhibit RAS-dependent MAPK activation. As FL–CNK, the first deletion construct (NT1271–CNK) also blocked RASV12 activity (Figure 3A, lane 4). In contrast and similar to NT–CNK, two other deletion constructs (NT1059- and NT659–CNK) no longer inhibited, but instead cooperated with RASV12 (Figure 3A, lanes 5–7). These data indicated that the C-terminal boundary of an inhibitory region, hereafter called RAF-inhibitory region (RIR), lies between amino acid position 1059 and 1271. Finer deletion constructs were then similarly tested, which positioned the RIR to a short area of ∼40 amino acids between positions 1059 and 1100 (see Supplementary figure S1, available at The EMBO Journal Online). Figure 3.Functional mapping of the RIR on CNK. (A) S2 cells were transfected with the indicated combinations of haMAPK (0.6 μg), haRASV12 (0.25 μg), FL and NT constructs (0.1–1 μg). Cell lysates were prepared 16 h post-induction of expression. (B) S2 cells were transfected with the indicated combinations of pyoRAF (0.7 μg), FL and CT constructs (1–1.8 μg) and NT construct (0.08 μg). Cells were lysed 36 h post-induction. For these experiments and below, pyoRAF was immunoprecipitated (IP) from cell lysates using the α-Pyo antibody and co-immunoprecipitated CNK proteins were detected using the α-Flag antibody. (C) S2 cells were transfected with the indicated combinations of pyoRAF (0.7 μg) and CT constructs (1–1.8 μg). (D) S2 cells were transfected with the indicated combinations of haMAPK (0.3 μg), haRASV12 (0.125 μg), NT constructs (0.6 μg). (E) Amino acid comparison of the Drosophila (D. mel) CNK RIR (pos. 1022–1100) to the equivalent region of A.gambiae (A. gam) CNK. Identical and conserved residues are in black and grey boxes, respectively. Positions of the ‘alanine-scanning’ mutations (M1–M12) are depicted as a solid line over the amino acid sequence. Minimal amino acid sequence for the RIM and the IS are also highlighted by a solid line over the relevant area. Although sequences within the 1022–1059 interval also appear to participate in RAF-binding (Supplementary figure S2B), these are not essential. Download figure Download PowerPoint As CNK associates with RAF, it could be responsible for the negative effect of CNK. To address this possibility, we mapped the RAF binding site(s) on CNK to determine whether it corresponds to the RIR. CT–CNK, but not NT–CNK, was previously found to interact with RAF (Therrien et al., 1998). We first tested whether we could reproduce these findings using a transient expression assay. A polyoma (pyo) epitope-tagged RAF construct was co-expressed with either FL–, NT– or CT–CNK in S2 cells. Cell lysates were immunoprecipitated using an anti-pyo antibody and co-immunoprecipitated CNK variants were detected by probing immunoblots with an anti-flag antibody. As shown in Figure 3B (lanes 2–4), FL– and CT–CNK, but not NT–CNK, associated with RAF (∼25% of RAF is associated with FL–CNK, and ∼50% of FL–CNK is associated with RAF in these conditions). Two C-terminal deletions of CT–CNK (CT-1 and CT-2, Figure 2A) were also included in that experiment. CT-1, which ended at position 1271, still bound to RAF, whereas CT-2, which ended at position 1059, no longer interacted (Figure 3B, lanes 5 and 6). These results thus placed the C-terminal border of the RAF-binding region, hereafter called the RAF-interacting motif (RIM), in the 1059–1271 interval. The finer C- and N-terminal truncations used above to map the RIR were then used to delineate more accurately the RIM. This analysis showed that sequences in the 1059–1077 interval are critical for RAF binding (see Supplementary figure S2). Finally, we narrowed down the RIM to a nine amino acid stretch (positions 1065–1073) by testing for RAF interaction, a series of ‘alanine scanning’ mutants within the 1059–1077 interval (M1–M6, Figure 3C and E). Together, our data thus revealed that the RIM is part of the RIR, which strongly suggests that the binding of RAF by CNK is responsible for the inhibitory effect of CNK. In support of this conclusion, the three point mutations (M3–M5) that impeded RAF binding (Figure 3C) also abrogated the inhibitory effect of CNK (data not shown and see below). Our mapping data showed that the RIR comprises additional sequences after the C-terminal end of the RIM (Figure 3E). This indicated that other sequences that are not required for RAF interaction have an inhibitory effect on RAS signaling. To define more precisely the position of these sequences, we tested a set of alanine scanning mutants within the 1077–1100 interval (M7–M12, Figure 3E) and found that mutants M7–M11 relieved the inhibitory effect of CNK (Figure 3D) but, as expected, did not prevent RAF binding (data not shown and see below). These results therefore confirmed that the RIR contains at least two distinct negative elements: the RIM that interacts with RAF and an adjacent inhibitory sequence (IS), which is required along with the RIM to inhibit signal transmission within the MAPK module. To verify whether the two negative elements (RIM and IS) also functioned accordingly in full-length CNK, we introduced the M4 mutation (affects the RIM) or the M11 mutation (affects the IS) in FL–CNK and examined their behavior with respect to RAS-induced MAPK activation and RAF-binding. Compared to FL–CNK (Figure 4A, lane 3), FLRIR(M4)–CNK and FLRIR(M11)–CNK no longer inhibited MAPK activation, but instead strongly cooperated with RASV12 (lanes 4 and 5). Furthermore, a double FLRIR(M4/M11)–CNK mutant cooperated to the same extent as either single mutants (lane 6), thus indicating that the two elements are co-required for the negative effect. As for NT–CNK (Figure 2), the ability of those mutants to cooperate with RAS appears to depend on the N-terminal domains of CNK as a double mutant version that affects both the SAM domain and the RIR barely cooperated with RASV12 (Figure 4A, lane 7). We next examined the ability of the M4 and M11 mutants to associate with RAF. As predicted, FLRIR(M4)–CNK no longer interacted with RAF (Figure 4B, lane 3), whereas FLRIR(M11)–CNK interacted normally with RAF (lane 4), thus confirming that only the RIM is essential for RAF-binding. Taken together, these results demonstrate that the RIM and IS elements, which constitute the RIR, are jointly required and sufficient to explain the inhibitory effect of CNK on the MAPK module. Figure 4.The negative effect of CNK is mediated by two co-required elements. (A) S2 cells were transfected with the indicated combinations of haMAPK (0.3 μg), haRASV12 (0.14 μg) and FL (0.5–0.8 μg) constructs. (B) S2 cells were transfected with the indicated combinations of pyoRAF (0.7 μg) and FL–CNK (1.5 μg) constructs. (C) S2 cells were transfected with the indicated combinations of haMAPK (0.3 μg), haRASV12 (0.2 μg) and GFP constructs (0.5 μg). Download figure Download PowerPoint Finally, to determine whether the RIR functions autonomously, we fused it (position 1059–1100) to the C-terminal end of GFP (GFP–RIR) and examined whether this was sufficient to transpose CNK's negative effect on GFP. As shown in Figure 4C, GFP–RIR strongly inhibited RAS-induced MAPK activation (lane 4), whereas GFP alone (lane 3) or two inactivated versions (lanes 5 and 6) of the RIR (RIRM4 or RIRM11) did not affect MAPK activation. These results therefore indicate that the RIR acts as an independent negative unit. The RIR of CNK antagonizes RAS signaling during eye development We wanted to determine whether the RIR of CNK also negatively influenced RAS signaling during Drosophila eye development. Intriguingly, in contrast to the data presented above, previous work showed that FL–CNK cooperated with RASV12 in the Drosophila eye (Therrien et al., 1998). We found, however, that this cooperation greatly depended on RASV12 signaling strength as well as FL–CNK expression levels, that is, FL–CNK inhibited RASV12 phenotype when a weaker RASV12 line was used (see below) or when FL–CNK levels were increased (data not shown). Nonetheless, the ability of FL–CNK to cooperate with RASV12 in the developing eye, a phenomenon not detectable in S2 cells by simply co-expressing various amounts of either protein, suggests that S2 cells might be missing a critical signal and/or factor. Although not mutually exclusive, another possibility is that there is a close relationship between the amount of activated RAS molecules and available N-te