1. Volume 8, Issue 5, Pages 983-993 (November 2001)
C-TAK1 Regulates Ras Signaling by Phosphorylating the MAPK Scaffold, KSR1 
Jürgen Müller, Stéphane Ory, Terry Copeland, Helen Piwnica-Worms, Deborah K. Morrison 
Molecular Cell 
Volume 8, Issue 5, Pages (November 2001)
DOI: /S (01)

2. Figure 1 Detection of KSR1-Associated Kinases
(A) Cos cells expressing either WT KSR1 or the various KSR1 deletion mutants were left untreated (−) or treated for 5 min with EGF (+) prior to lysis. The KSR1 proteins were immunoprecipitated from the lysates using Pyo antibody, and immune complex kinase assays were performed. Labeled proteins were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography (left panel). The membrane was then probed with Pyo antibody to verify the expression of the transfected proteins (right panel) and reprobed with antibodies against MAPK and MEK (lower left panels). WT KSR1 and the deletion mutants are schematically depicted.
(B) KSR1 immunoprecipitates were prepared as in (A), and immune complex kinase assays were performed with the addition of purified Raf-1. The membrane was probed with a Raf-1 antibody to verify that equal amounts of Raf-1 were present in the reactions and reprobed with an antibody recognizing phosphorylated and activated MEK (P-MEK).
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 The N′424-Associated Kinase Phosphorylates KSR1 at S392
KSR1 proteins labeled in immune complex kinase assays were digested with trypsin, and the tryptic phosphopeptides were separated by HPLC. The profile of radioactivity collected in the HPLC column fractions is shown for WT KSR1, N′424, and N′424 proteins containing serine to alanine mutations at amino acid residues 297 (S297A) and 392 (S392A). The number under the Edman degredation column indicates the cycle in which 32P counts were released. The asterisk indicates the residue phosphorylated.
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3 C-TAK1 Associates with KSR1
(A) N′424 and C′542 proteins were immunoprecipitated from cycling Cos cells, and the immune complexes were examined by immunoblot analysis using antibodies recognizing C-TAK1, Akt, PKC, Chk1, and the Pyo epitope. Total cell lysate was included as a control to identify the position of the respective protein (arrow). The migration of the IgG heavy chain is indicated by an asterisk.
(B) KSR1 deletion mutants and FLAG-epitope-tagged proteins encoding the N-terminal and C-terminal domains of Raf-1 were immunoprecipitated from Cos cells, and the immune complexes were examined by immunoblot analysis using C-TAK1, Pyo, and FLAG antibodies.
(C) KSR1 proteins were immunoprecipitated from untreated (−) and EGF-treated (+) Cos cells. KSR1, Raf-1, and rabbit IgG immunoprecipitates were also prepared from mouse brain lysates. The immune complexes were then examined by immunoblot analysis using C-TAK1, KSR1, Raf-1, and Pyo antibodies.
(D) N′424 and N′424 proteins containing alanine substitutions at amino acid residues 397 and 401 (N′424 IV/AA) were immunoprecipitated from cycling Cos cells, and immune complex kinase assays were performed. The labeled proteins were examined by autoradiography and immunoblot analysis using C-TAK1 and Pyo antibodies.
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 In Vitro and In Vivo Phosphorylation of KSR1 on S392
(A) N′424 and C′542 KSR1 proteins were incubated with recombinant C-TAK1, Akt, Chk1, and Chk2 in the presence of [γ-32P]ATP. Phosphorylated KSR1 proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and quantitated using a phosphoimager. The activities of Chk1 and Chk2 were confirmed using the CHKtide substrate, and the activity of Akt was demonstrated using the Crosstide substrate (data not shown).
(B) N′424 phosphorylated by C-TAK1 in vitro was digested with trypsin and examined by HPLC analysis.
(C) Cos cells expressing WT or S297A/S392A KSR1 were labeled in vivo with [32P]orthophosphate and either left untreated or were treated with EGF prior to cell lysis. KSR1 proteins were immunoprecipitated, digested with trypsin, and examined by HPLC analysis. The values for the cpm incorporated into the S392 site are 12,461 cpm from untreated cells and 6,017 cpm from EGF-treated cells.
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
Effect of S392 Mutation on KSR1 Subcellular Localization, Complex Formation, and Biological Activity
(A) Cos cells expressing WT, S392A, or S297A/S392A KSR1 were left untreated or were treated with EGF. The intracellular localization of the KSR1 proteins was then determined by indirect immunofluorescence using Pyo antibody.
(B) KSR1 proteins were immunoprecipitated from untreated (−) or EGF treated (+) Cos cells, and the immune complexes were examined by immunoblot analysis using Pyo, MEK, MAPK, and antibodies.
(C) Xenopus oocytes were injected with RNA encoding either the WT, S392A, or S297A/S392A KSR1 proteins and activated RasV12. Germinal vesicle breakdown (GVBD) was scored 5 hr following Ras injection. Oocyte lysates were prepared and examined by immunoblot analysis using Pyo and phospho-MAPK antibodies (P-MAPK).
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 Colocalization of KSR1, MEK, and Activated MAPK
Cos cells expressing WT, S392A, or S297A/S392A KSR1 were left untreated or were treated with EGF. The intracellular localization of the KSR1 proteins (A and B), MEK (A), and activated MAPK (B) was determined by indirect immunofluorescence using Pyo, MEK, and phospho-MAPK (P-MAPK) antibodies.
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 Model for KSR1 Function
KSR1 is localized in the cytoplasm of quiescent cells with MEK bound to its kinase-like domain and a dimer of bound to the S297 and S392 phosphorylation sites. C-TAK1 constitutively associates with the N-terminal region of KSR1 to maintain the phosphorylation of S392 and, consequently, the binding of to this site (the regulated site, “Reg”). In response to input signals such as growth factor treatment, the S392 site of KSR1 becomes dephosphorylated (by an unknown mechanism), perhaps exposing the KSR1 C1 domain and the FxFP MAPK binding site. As a result, the KSR1 complex translocates to the plasma membrane, colocalizing MEK with its upstream activator Raf-1 and downstream target MAPK.
Molecular Cell 2001 8, DOI: ( /S (01) )