Fig. 1: Rmuts and Cmuts exhibit different biochemical and biological properties.
a Schematic diagram of MEK1 mutations in primary cancers (red) and RASopathies (blue). b HEK293 cells were transfected with wild type or mutated HA-MEK1 together with kinase-deficient Myc-ERK2(K/N) as indicated. Phosphorylated ERK (P-ERK) was detected by immunoblotting with an anti-phospho-ERK antibody (Ab) (top). Myc-ERK2(K/N) and HA-MEK1 expression levels are shown in the lower panels. MEK1(DD); a phosphomimetic, constitutively active mutant. c In vitro kinase assay of purified recombinant GST-MEK1 or its mutant derivatives using GST-ERK2(K/N) as a substrate. Phosphorylated ERK2(K/N) was detected by immunoblotting (top). Total GST-MEK1 and GST-ERK2(K/N) were also probed with an anti-GST Ab (bottom). d, e MEK1−/− MEFs stably expressing HA-MEK1 or its indicated mutants were cultured in medium with 10% FBS (d) or were grown in soft agar for 3 weeks (e). In (d), cell proliferation was determined by CCK8 assay. OD, optical density. In (e) colonies larger than 0.1 mm were counted. f, g, j, k HA-MEK1 (WT or its indicated mutants) expressed in HEK293 cells (f, k) or bacterially expressed, purified GST-MEK1 (WT or its indicated mutants) (g, j) was immunoblotted with an anti-phospho-MEK1(S218/S222) Ab (P-MEK). Raf-1ΔN; N-terminally truncated, active Raf-1. K/M; a kinase-inactive K97M mutation. h Recombinant GST-MEK1 proteins were pre-incubated with (+) or without (−) active His-BRafV600E, and then mixed with GST-ERK2(K/N). Phosphorylated ERK (P-ERK) and phosphorylated MEK1 (P-MEK) were detected by immunoblotting. AA; a non-phosphorylatable S218A/S222A mutant. i HEK293 cells were transfected with HA-MEK1 or its indicated mutants and were stimulated with (+) or without (−) TPA (for 20 min). Phosphorylated HA-MEK1 in cell lysates was monitored by immunoblotting (third). The kinase activity of immunoprecipitated HA-MEK1 was measured in an in vitro kinase assay using GST-ERK2(K/N) as a substrate as in h. l The indicated GST-MEK1 proteins were incubated with GST-ERK2(K/N), and the phosphorylation states of MEK1 and ERK2 were assessed by immunoblotting. d, e Data are mean ± SEM from three independent experiments. P-values were assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (d) or using two-tailed Student t-test (e). Source data are provided as a Source Data file.
