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Figure 1 cPLA 2 Binds to KSR1 (A) cPLA 2 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.
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(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) cPLA 2 levels present in total lysates and in anti-ERK2 immunoprecipitates. (Left panel, bottom) Endogenous cPLA 2 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 cPLA 2 is triggered by EGF stimulation and is inhibited by UO126 (2 μM, 25 min prior stimulation). (D) cPLA 2 does not directly bind to KSR1.
(Top panel) The presence of KSR1 was analyzed in anti-cPLA 2 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 cPLA 2. TL, total lysate. Immunoprecipitations were performed with a specific antibody (IP) or with preimmune serum (PI). Figure 2 KSR1 Binds to ERK2 Dimers (A) ERK2 binds to cPLA 2 through a hydrophobic region −FXF interaction. FLAG-tagged ERK2, wild-type and Y261A (1 μg each), was transfected in 293T cells.
The presence of cPLA 2 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.