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. 2023 Apr 12;299(5):104699. doi: 10.1016/j.jbc.2023.104699

Figure 3.

Figure 3

MK2 catalyzes RSK1 phosphorylation at Ser-380.A, HeLa cells were treated with 10 μM MK2 inhibitor III (MK2 inh III) for 30 min and then stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. B, HeLa cells were transfected with siRNA against MK2 or the negative control. At 48 h post-transfection, cells were stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. A and B, Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, phospho-MK2 (pMK2), and α-Tubulin. Red arrow, phosphorylated MK2; blue arrow, non-phosphorylated MK2. C, HEK293 cells were transfected with expression vectors for EGFP-tagged kinase-dead EphA2 (EphA2-KD-EGFP), FLAG-tagged RSK1, FLAG-tagged p38α, MK2 (wild-type (WT) or kinase-dead mutant (KD)), and/or an empty vector. At 24 h post-transfection, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, FLAG, MK2, and β-Actin. D, HeLa cells were treated with 10 μM MK2 inhibitor III and then stimulated with 100 μM CDDP for 3 h or 0.3 M NaCl (Osmo) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, pMK2, and α-Tubulin. E, immunoprecipitated RSK1 and RSK2 prepared from HeLa cells were incubated with recombinant human active GST-MK2 at 30 °C for 30 min. Reaction mixtures were analyzed by immunoblotting with anti-phospho-RSK (Ser-380 of RSK1; Ser-386 of RSK2), RSK1, RSK2, and pMK2 antibodies. EphA2, ephrin type-A receptor 2; MK2, MAPK-activated protein kinase 2, RSK, p90 ribosomal S6 kinase.