1. Essential Role of ERK Dimers in the Activation of Cytoplasmic but Not Nuclear Substrates by ERK-Scaffold Complexes 
Berta Casar, Adán Pinto, Piero Crespo 
Molecular Cell 
Volume 31, Issue 5, Pages (September 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 cPLA2 Binds to KSR1
(A) cPLA2 activation by EGF (100 ng/ml, 5 min) is downregulated by depletion of scaffold proteins. 293T cells were transfected with siRNAs (10 ng) for the shown scaffold proteins. A set of four random, scrambled siRNAs (SCR) was utilized as control. (Right panel) Diminished expression of endogenous scaffold proteins by siRNA treatment. For dystroglycan and Sef, Myc-tagged proteins were used. (Left panel, top) Phosphorylated and total ERK levels and total and phosphorylated (S505) cPLA2 levels present in total lysates and in anti-ERK2 immunoprecipitates. (Left panel, bottom) Endogenous cPLA2 activation determined by [3H] AA release. Results show average ± SEM of five independent experiments relative to starved (st) cells.
(B) ERK2-KSR1 association is enhanced upon stimulation. Endogenous ERK2 presence in native KSR1 immunoprecipitates was determined in cells starved or treated with EGF.
(C) The association of KSR1 and endogenous cPLA2 is triggered by EGF stimulation and is inhibited by UO126 (2 μM, 25 min prior stimulation).
(D) cPLA2 does not directly bind to KSR1. (Top panel) The presence of KSR1 was analyzed in anti-cPLA2 and anti-MEK immunoprecipitates from starved 293T cells. (Bottom panel) In vitro-translated KSR1 (INPUT) is pulled down (PD) by bacterially produced MEK but not by cPLA2. TL, total lysate. Immunoprecipitations were performed with a specific antibody (IP) or with preimmune serum (PI).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 KSR1 Binds to ERK2 Dimers
(A) ERK2 binds to cPLA2 through a hydrophobic region −FXF interaction. FLAG-tagged ERK2, wild-type and Y261A (1 μg each), was transfected in 293T cells. The presence of cPLA2 was assayed in anti-FLAG immunoprecipitates in cells starved or treated with EGF (100 ng/ml, 5 min).
(B) Endogenous KSR1 binds to ERK2 dimers. Total and phosphorylated ERK2 species in endogenous KSR1 immunoprecipitates were examined in 293T cells starved, EGF treated, or transfected with H-RasV12 (1 μg).
(C) KSR1 binds to ERK2 dimers in MDCK cells. The forms of ERK2 in endogenous KSR1 immunoprecipitates were determined in MDCK cells starved or treated with EGF or LPA (5 μM, 5 min). FPLC-purified ERK monomers and dimers were included as markers. A lysate from 293T cells was included to compare in both cell types the sizes of the bands with different mobilities.
(D) ERK2 wild-type, but not HL, dimerizes. HA-tagged ERK2, wild-type and HL (1 μg each), was transfected into 293T cells, and ERK species were examined by anti-HA immunoblotting in total lysates from cells starved or treated with EGF.
(E) ERK2 Y261A binds to KSR1 by dimerization. Cells were trasfected with FLAG-tagged ERK2 wild-type or Y261A, alone or with HA-ERK2 HL (1 μg each). Their presence in anti-KSR1 immunoprecipitates was determined by anti-FLAG imunoblotting in cells starved or stimulated with EGF.
(F) ERK2 HL diminishes the amount of ERK dimers. The forms of endogenous ERK2 were determined in total lysates from proliferating 293T cells, parental or transfected with HA-ERK2 HL (1 μg).
(G) Increasing concentrations of ERK2 HL (0.2–2 μg) displace endogenous ERK2 monomers from KSR1 immunoprecipitates in starved cells.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 ERK Dimers Mediate in cPLA2-KSR1 Interaction
(A) Effects of increasing concentrations of HA-tagged ERK2 HL (0.2–2 μg) in the EGF-induced association to KSR1 of ERK2 dimers, endogenous cPLA2, and MEK. Phosphorylated ERKs levels in total lysates are shown at the bottom.
(B) ERK2 HL is impaired for binding to cPLA2. As determined in anti-HA immunoprecipitates from cells transfected with HA-tagged ERK2, wild-type or HL (1 μg each), starved or stimulated with EGF.
(C and D) Increasing concentrations of (C) ERK2 HL (0.5–2 μg) and (D) ERK2 Y261A (1–2 μg) diminish EGF-induced activation of cPLA2. Determined by [3H] AA release and by analyzing total and phosphorylated cPLA2 in anti-ERK2 immunoprecipitates. Results show average ± SEM of three experiments relative to starved cells.
(E) ERK cytoplasmic substrates bind to ERK2 in dimeric form. Endogenous cPLA2, RSK1, and PDE-4 were immunoprecipitated in 293T cells, starved or EGF stimulated. The associated ERK2 species were examined by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Scaffold Proteins in General Bind to ERK Dimers
(A) The association between Sef and ERK2 is enhanced by EGF stimulation (100 ng/ml, 5 min). Cells were transfected with Myc-tagged Sef (1 μg), and the presence of ERK2 was assayed in anti-Myc immunoprecipitates.
(B) The association of Sef and cPLA2 is induced by EGF and inhibited by UO126.
(C) Sef binding to ERK dimers is inhibited by ERK2 HL. Cells were transfected with Myc-Sef (1 μg). The forms of ERK2 were analyzed in anti-Myc immunoprecipitates in cells starved or treated with EGF in the absence or presence of ERK2 HL (1 μg).
(D) Increasing concentrations of HA-ERK2 HL (0.5–2 μg) prevent EGF-induced endogenous cPLA2 binding to Sef.
(E) Endogenous IQGAP and Paxillin associate to ERK2 dimers in response to EGF.
(F) MP-1 and MORG bind to ERK1 dimers. Cells were transfected with Myc-MP1 or FLAG-MORG plus HA-ERK1 (1 μg each). The formation of dimers was assayed by anti-HA immunoblotting in anti-Myc or -FLAG immunoprecipitates after treatment with EGF (MP-1) or LPA (MORG).
(G) ERK2 HL prevents the formation of ERK1 dimers in KSR1. Cells were transfected with FLAG-ERK1, alone or with HA-ERK2 HL (1 μg each). The formation of dimers was monitored by anti-FLAG immunoblotting in anti-KSR1 immunoprecipitates after EGF treatment.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Scaffolds and ERK Dimers Are Not Involved in the Activation of Nuclear Substrates
(A) Depleting scaffold proteins by siRNAs enhances Elk1 activation induced by EGF (100 ng/ml, 5 min). Elk1 transactivation was examined in 293T cells transfected with GAL4-Elk1 (TAD) (1 μg) plus siRNAs (10 ng) for the shown scaffolds. Total and phosphorylated Elk1 levels were determined by immunoblotting.
(B) Inhibiting ERK dimerization potentiates Elk1 transactivation and phosphorylation induced by EGF, as shown in cells starved or EGF stimulated, in the absence or presence of HA-ERK2 HL (1 μg). (A and B) Results show average ± SEM of three independent experiments relative to starved cells.
(C) (Top panel) ERK2 HL potentiates ERK nuclear entry. The levels of endogenous ERK2 in nuclear extracts from control and ERK2 HL (1 μg)-transfected cells were monitored after treatment with EGF for the indicated times. (Bottom panel) ERK2 HL does not enter the nucleus. The levels of HA-ERK2 HL in cytoplasmic and nuclear extracts were monitored upon stimulation with EGF.
(D) ERK2 is translocated into the nucleus mainly as monomers, as determined in cytoplasmic and nuclear fractions from cells treated with EGF for the indicated times. (Top panel) The purity of the nuclear and cytoplasmic fractions was ascertained using Elk1 and Rho-GDI as markers.
(E) Mxi2 translocates ERK2 into the nucleus mainly in as monomers. Cells were transfected with a Zn-inducible Mxi2 expression plasmid (1 μg) and stimulated with ZnSO4 for the indicated times.
(F) Nuclear substrates bind to ERK2 monomers. Endogenous Elk1, Fos, and Fra1 were immunoprecipitated in 293T cells, starved or EGF-stimulated, and the associated ERK2 species were examined by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
Preventing ERK Dimerization Inhibits Cellular Transformation, Proliferation, and Tumor Development
(A) ERK2 HL inhibits transformation. (Top panel) Nucleocytoplasmic segregation of ERK2, HL, and HL-NLS, transfected in 293T cells (1 μg). The purity of the fractions was ascertained using Elk1 and Rho-GDI. (Lower panel) Focus formation assays in NIH 3T3 cells, transfected with the shown oncogenes (0.25 μg) plus ERK2, HL, or HL-NLS. Results show average ± SEM of three independent experiments. ∗∗∗p < ; NS, p > 0.05 with a 95% confidence interval.
(B) ERK dimerization is impaired in tumor cell lines expressing ERK2 HL. ERK2 species and total ERK phosphorylation levels were analyzed in HCT116 cells: parental (par) and stably expressing ERK2, HL, or HL-NLS. Immunoblotting revealed the levels of total and phosphorylated RSK1 and Elk1 in total lysates and of cPLA2 present in anti-ERK2 immunoprecipitates.
(C) Proliferation kinetics of HCT116, T24, and H1299-derived cell lines: parental (blue), ERK2 HL (green), and ERK2 HL-NLS (red).
(D) Tumor development after 3 weeks in nude mice injected with HCT116-derived cell lines expressing the ERK2 dimerization mutants: parental (left), ERK2 HL-NLS (middle), and ERK2 HL (right).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7 A Model for the Role of Scaffold Proteins in ERK Signaling
Under resting conditions, ERKs exist in two types of complexes. In response to agonists, phosphorylated ERK monomers detach and may follow three destinies: (1) translocate as monomers to the nucleus, where they activate transcription factors; (2) dimerize freely in the cytoplasm; free dimers could remain in the cytoplasm or be transported into the nucleus; or (3) bind ERK monomers already attached to the scaffold complexes to form a dimer whereby the scaffolded complex can interact with cytoplasmic substrates. Cyt. Subs, cytoplasmic substrates; Tr. Factor, transcription factors.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions