Model accounting for KSR function. See text for details.

KSR enhances MEK phosphorylation by RAF in a RAS-dependent manner. (A) S2 cells were transfected with the mycMEK^DA reporter construct (0.3 μg) either alone (lane 1) or together with the indicated combinations of RAS^V12 (0.6 μg), KSR (0.1 μg), and pyoRAF (0.03 μg) constructs. Cells were lysed 36 h postinduction of expression, and pMEK levels were examined by immunoblot analysis. Protein levels were evaluated for KSR, pyoRAF, and mycMEK^DA as indicated. (B) S2 cells were transfected as in A with the indicated combinations of expression plasmids. KM denotes pyoRAF^K498M or KSR^K705M.

KSR is a bona fide component of the MAPK pathway. (A) Untreated (−) or CuSO₄-treated (+) RAS^V12 cells were either incubated alone (−) or with (+) 10 μg of the indicated dsRNAs. After incubation, cells were lysed and pMEK or pMAPK levels, or protein levels for RAS1, RAF, MEK, and MAPK, were analyzed by immunoblot analysis using the indicated antibodies. Endogenous KSR protein levels were under the detection limit of our KSR antibody. Immunoprecipitation from ∼25× more cells is required to detect endogenous KSR, which is incompatible with the RNAi protocol. (B) S2 cells were transfected with pMet-KSR and were either untreated (−) or CuSO₄-treated (+) to induce KSR expression. Cotransfection of pMet-KSR with 0.5 μg of dsKSR RNA prevented KSR accumulation, thus showing the effectiveness of dsKSR RNA.

KSR activity depends on multiple regions of the KSR protein. (A) Structure of KSR and amino acid positions of various mutants used in this study. In addition to a putative kinase domain, the conserved area 1 (CA1), the proline-rich stretch (Pro), the cysteine-rich motif (CRM), and the serine/threonine-rich stretch/FXFP motif (S/T FXFP) are other features of the KSR proteins (Therrien et al. 1995). (B) S2 cells were transfected with the indicated combinations of mycMEK^DA (0.6 μg), RAS^V12 (0.6 μg), pyoRAF (0.2 μg), and wild-type (WT) KSR (0.7 μg) or one of the indicated KSR mutants (0.7 μg). Cells lysates were prepared 36 h postinduction of expression and either directly used to determine pMEK levels or immunoprecipitated with a monoclonal anti-KSR antibody to examine the amounts of mycMEK^DA and pyoRAF associated with KSR. Protein levels for KSR, mycMEK^DA, and pyoRAF were also evaluated.

KSR interacts independently with RAF and MEK and induces the formation of a RAF/MEK complex. (A) S2 cells were transfected with pyoRAF alone or together with KSR in the presence of the indicated dsRNAs (0.5 μg; lanes 2–5). Cell lysates were prepared and immunoprecipitated 36 h postinduction as indicated. Levels of associated proteins and their relative amounts were evaluated by immunoblot analysis. (B) S2 cells were transfected with mycMEK^DA (0.2 μg) and pyoRAF (0.2 μg) constructs either alone (lane 3) or with increasing quantities for wild-type (WT) KSR (0.02 μg, 0.08 μg, and 0.325 μg) or for KSR^L50S-R51G or KSR^C922Y (0.02 μg, 0.08 μg, 0.325 μg, and 1.3 μg), as indicated; control (lane 1) used 1.3 μg of wild-type KSR. Cell lysates were prepared 36 h postinduction and immunoprecipitated by the anti-pyo antibody to measure associated MEK and KSR levels by immunoblot analysis. Protein levels in lysates were also determined. (C) S2 cells were transfected with mycMEK^DA (0.2 μg), pyoRAF (0.2 μg), or KSR (0.7 μg) without or with 0.6 μg of HA-epitope-tagged RAS^V12 or 0.5 μg of dsRAS1 RNA as indicated. Cell lysates were prepared as in C. ha-RAS^V12 levels were examined using anti-HA antibody. Note that addition of dsRAS1 RNA eliminates ha-RAS^V12 completely.

TorKSRc, a dominant-negative form of KSR, strongly cooperates with TorRAFc, an activated form of RAF, to activate the MAPK pathway during Drosophila eye development. (A,C,E,G) Anti-pMAPK staining of Drosophila third instar eye imaginal discs and (B,D,F,H) scanning electron micrographs of adult Drosophila eyes of the following genotypes: (A,B) wild type (WT); (C,D) BT98/+; (E,F) KDN/+; (G,H) BT98/KDN. Anterior is to the right. During normal eye disc development (A), activation of MAPK first appears in groups of cells in the morphogenetic furrow (arrowhead). Posterior (left) to the morphogenetic furrow, pMAPK staining is restricted to individual cells undergoing cell fate specification. The RAFc and KSRc constructs have been expressed under the sevenless enhancer sequences, which drives transgene expression in a subset of cells posterior to the morphogenetic furrow: arrow in A marks approximately the rows of ommatidial clusters where transgene expression occurs.

Induced association between KSR and RAF catalytic domains triggers MEK phosphorylation. (A) Schematic of the FKBP–RAF and FRB–KSR fusion proteins used in this study. (B) S2 cells were transfected with mycMEK^DA (1 μg) alone or with the indicated combinations of KSRc (0.2 μg) and RAFc (0.3 μg) constructs. Twenty-four hours postinduction of expression, cells were treated with either vehicle (ethanol; −rapamycin) or with 1 μM rapamycin (+rapamycin) for 2 h prior to harvest. Cell lysates were prepared, and small fractions were used to evaluate pMEK and mycMEK^DA levels by immunoblot analysis. The remaining portion of the lysates was split and immunoprecipitated with either anti-pyo or anti-Flag antibody to monitor RAFc or KSRc protein levels, associated mycMEK^DA, and heterodimerization with the opposite FRB or FKBP fusion proteins. Heterodimerization data are not shown, for simplicity. The results shown here and in Figures 3–6 are representative of at least three similar experiments. (C) The experiments were conducted essentially as described in B to evaluate the ability of myr-FKBP–RAFc^K498M and myr-FRB–KSRc^K705M to induce MEK phosphorylation. Because of the posttranslational modification, the myristoylated fusion proteins show faster mobility. We noticed that the dimerization of different fusion proteins was less rapamycin-dependent when they were either both membrane-targeted or not. This was presumably caused by the tendency of these proteins to associate together. However, dimerization was strictly rapamycin-dependent when RAFc proteins were membrane-targeted, but not the KSRc proteins or vice versa. Therefore, we presented only the data for which either RAFc or KSRc is membrane-targeted.