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. Author manuscript; available in PMC: 2011 Feb 22.
Published in final edited form as: Nat Cell Biol. 2010 Nov 21;12(12):1242–1249. doi: 10.1038/ncb2130

Figure 5.

Figure 5

Phosphorylation of KSR-1 on Ser 838 controls ERK1/2 signalling. (a) KSR-1 immunoprecipitates from KSR-1−/− mouse embryonic fibroblasts (MEFs) and MEFs expressing wild-type KSR-1 were screened for AKAP-Lbc, PKA RII and KSR-1 by immunoblotting. (b) Time-course of EGF-stimulated ERK1/2 activity in KSR-1−/− MEFs and MEFs expressing wild-type KSR-1 or KSR-1S838A. Starved cells were treated with EGF for the indicated times. ERK activation was assessed by immunoblotting for phosphorylated ERK1/2, ERK1/2, KSR-1 and AKAP-Lbc. (c) Quantification of phosphorylated ERK1/2 bands by densitometry, from experiment carried out as in b (data are means ± s.e.m.). (d) Time-lapse microscopy images of FRET signals in HEK293 cells expressing EKAR, and AKAP-Lbc and wild-type KSR-1 (top), or AKAP-Lbc and KSR-1S838A (bottom), at indicated times after addition of EGF (min). Scale bars, 20 μm. (e) Quantification of YFP/CFP ratios for experiment performed as in d. Black bar indicates addition of EGF. Data are means ± s.e.m. (f) HEK293 cells expressing wild-type KSR-1 or KSR-1S838A were starved and stimulated with forskolin/IBMX, as indicated. ERK activation was measured by immunoblotting of the cell lysates. (g) Quantification of phosphorylated ERK1/2 bands by densitometry from experiments performed in f. Data are normalized to the density of the total ERK1/2 bands. Data are means ± s.e.m. (asterisk indicates P < 0.05, paired t-test). (h) KSR-1−/− MEFs and MEFs expressing wild-type or KSR-1S838A were treated with forskolin and IBMX, and H-89, as indicated. ERK activation was assessed by immunoblotting for phosphorylated ERK1/2, ERK1/2, KSR-1 and AKAP-Lbc. (i) Quantification of phosphorylated ERK1/2 bands by densitometry from experiments performed in h. Data are normalized to the density of the total ERK1/2 bands. Data are means ± s.e.m. (asterisk indicates P < 0.001, ANOVA). (j–u) Immunofluorescence microscopy analysis of ERK activation. KSR-1−/− MEFs (j–m), and MEFs expressing KSR-1 (n–q) or KSR-1S838A (r–u) were starved, treated with forskolin/IBMX for 10 min, fixed, and immunostained for phosphorylated ERK1/2 and total ERK1/2, as indicated. Scale bars, 20 μm. (v) Schematic representation of the AKAP-Lbc–KSR-1 core unit directing growth factor and cAMP signals through the ERK signalling network. Uncropped images of blots are shown in Supplementary Information, Fig. S7.