The N-terminal portion of CNK augments KSR-induced MEK phosphorylation by RAF. (A) S2 cells were transfected using the indicated plasmid combinations. RAF activity was assessed by determining phospho-MEK levels in cell lysates by immunoblotting (α-pMEK). Protein levels were verified using the antibodies indicated to the right. Results presented here and thereafter are representative of at least three similar experiments. RAF and KSR kinase-mutant (KM) variants have a K455S and a K705M change, respectively. As previously reported (Douziech et al. 2003), coexpression of full-length CNK along with RAS^V12 is inhibitory. This is caused by the RIR and is naturally alleviated by RTK-induced Src42 binding to the Y1163 region located C-terminal to the RIR (Laberge et al. 2005). To facilitate the characterization of the positive effect of the SAM and CRIC domains with respect to RAS-dependent RAF activation, we used NT-CNK constructs instead of full-length CNK, thereby bypassing the requirement in additional RTK signals. The SAM^mut NT-CNK variant has a L71K mutation, while the CRIC^mut variant has a three-amino-acid deletion (Del A162-H163-R164) that is similar to a mutation found in a cnk loss-of-function allele (Therrien et al. 1998). The difference between the effects produced by the mutations within the SAM domain and the CRIC region may be due to the fact that the CRIC mutation is hypomorphic, while the L71K mutation obliterates normal SAM domain function. Alternatively, the involvement of the CRIC region may not be as decisive as the SAM domain. (B) Schematic of the main Drosophila RAF, KSR, and CNK constructs used in this study. Full-length RAF contains an RBD, a CRD, and a kinase domain (black box). The relative positions of the S346 (S259-like), K455 (critical lysine in subdomain II), and T571–T574 (phospho-accepting sites in the activation loop) residues are also shown. Full-length KSR comprises the so-called conserved area 1 (CA1) (Therrien et al. 1995), a CRD, and a putative kinase domain (black box). The conserved lysine residue of subdomain II (K705) and the C922 residue that is critical for MEK binding (Roy et al. 2002) are also represented. Schematic of the N-terminal (1–655) and C-terminal (656–1003) KSR constructs are indicated as tick lines. Full-length CNK includes a SAM domain, a CRIC, a PDZ domain, a proline-rich strech (Pro), a PH domain, and a RIR that includes two jointly required elements: a RIM and an IS. NT-CNK (position 2–384), NT⁵⁴⁹-CNK (position 2–549), and CT-CNK (position 382–1557) constructs are depicted as thick lines.

CNK activity is KSR-dependent. (A,B) S2 cells were transfected using the indicated plasmid combinations. dsRNA (500 ng) targeting the 3′UTR of KSR was also included in each condition. The CA1^mut amino acid change corresponds to L50S–R51G.

CNK mediates the formation of a KSR/RAF complex. (A–C) S2 cells were transfected using the indicated plasmid combinations and cell lysates were either directly probed or immunoprecipitated as indicated. The star in A denotes the position of a nonspecific protein revealed by the α-Flag antibody. (D) S2 cells were plated with the indicated dsRNAs (10 μg/mL). Four days later, cells were lysed and protein extracts were immunoprecipitated using an α-KSR monoclonal antibody. Two 100-mm dishes were pooled for each condition except for lane 7, where five dishes had to be pooled to obtain equal KSR protein levels, as depletion of endogenous MEK greatly destabilized KSR (not shown). (E) S2 cells were incubated with or without dsRNA (RNAi; 10 μg/mL) directed at CNK 3′UTR sequences. Cells were transfected 24 h later using the indicated combinations of plasmids (+500 ng of CNK 3′UTR dsRNA) and were processed as indicated.

Model summarizing the scaffolding and the RTK-dependent activating property of the KSR/HYP/CNK complex with respect to RAF and MEK. For simplicity, the model (detailed in the text) does not include the presumed effects of 14–3–3 protein binding on RAF and KSR conformation and localization. Dotted arrows represent possible entry points for RAS activity that is integrated by the complex independently of the RAF RBD. The arrow with a question mark between KSR and RAF kinase domains illustrates the putative RAS-dependent RAF-activating effect of KSR in association with CNK/HYP.

CNK and KSR function upstream of the RAF activation loop phosphorylation event. S2 cells were transfected using the indicated plasmid combinations. (A–C) In each condition, dsRNA (500 ng) targeting the 3′UTR of endogenous RAF was also transfected. The RBD^mut, CRD^mut, AL^ED, and AL^AA amino acid changes correspond to R174L, C249S–C252S, T571E–T574D, and T571A–T574A, respectively. (D) S2 cells were plated with or without RAF 3′UTR dsRNA (10 μg/mL) in combination with the other indicated dsRNAs (RNAi; 10 μg/mL each) and cultured for 24 h prior to transfection (500 ng of the same dsRNAs was also added to the transfection mixture). RAF activity was evaluated by determining phospho-MAPK levels. GFP dsRNA is used as a negative control for RNAi. Addition of NT-CNK further reduced the mobility of RAF (cf. lanes 3 and 4, α-PYO in A). This event is most likely due to phosphorylation, as the mobility shifts could be eliminated by in vitro phosphatase treatment (data not shown). Although the phosphorylation sites and their functional relevance are unknown, there is a tight correlation between the positive effects of NT-CNK and RAF mobility shift, which we used as a second readout of NT-CNK effect on RAF.

Two inactive KSR mutants retain their ability to associate with RAF and MEK. S2 cells were transfected using the indicated plasmid combinations. (A,B) Cell lysates were immunoprecipitated using an α-V5 antibody to determine the amounts of MEK or RAF associated to KSR. Lysates were also directly probed to monitor protein levels. (C) RAF immunoprecipitation (α-PYO) was used to verify the capacity of KSR^{A696V–A703T}, compared with wild-type KSR, to promote the formation of a RAF/MEK complex.

KSR recruitment to CNK depends on Hyphen, a novel SAM domain-containing protein. (A) Depletion of endogenous HYP by RNAi reduces MAPK activation (pMAPK levels) induced by RAS^V12, but not by RAF-AL^ED. (B) HYP is required for MEK phosphorylation stimulated by NT-CNK. R-R-K-M denotes the cotransfected RAS^V12, RAF, KSR, and MEK^DA plasmids. S2 cells in lanes 3–5 were plated with either dsGFP or dsHYP 3′UTR RNAs (6 μg/mL) for 24 h prior to transfection (500 ng of the same dsRNAs was included in the transfection mixture). (C) HYP is required for the CNK/KSR association. RNAi treatment was conducted as in B. (D,E) HYP associates with CNK independently of KSR and requires the integrity of the SAM domain of CNK. A PYO-tagged HYP (Δ3′UTR) variant was used for these experiments as the AU5-tagged variant did not immunoprecipitate quantitatively. PYO-HYP also fully rescued MEK activation and CNK/KSR interaction following depletion of endogenous HYP (not shown). (E) Endogenous HYP mediates KSR/CNK complex formation in vivo. The experiment was conducted as in Figure 5D.