Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells
Fig 5
Nuclear YopM controls phosphorylation of nuclear RSK1.
A) Coexpression of HA-RSK1 and myc-YopM increases phosphorylation of nuclear RSK1. (Left panel) HEK293T cells were transfected with indicated expression vectors and cytosolic- (C) and nuclear (N) fractions were investigated by immunoblot using indicated antibodies. Anti-pS221- and Anti-pS380 antibodies detect the respective phosphorylated amino acids in RSK1. Exposure time of the anti-RSK1 panel in the HA-RSK1 expressing cells (right, WB 1) is about 1/5 of that of the respective panel in control cells (left). (Right panel) Intensities of the pS221- pS380- and RSK1 protein bands from WB 1 and WB 2, the latter derived from a second identical experiment were quantified and intensity ratios of pS221/RSK1 and pS380/RSK1 were calculated. B) Leptomycin B treatment and DDX3 knockdown increase phosphorylation of nuclear RSK1. (Left panel) HEK293T cells were not treated (Ctrl) or treated with 25 nM LMB for 4 h. (middle and right panels) HEK293T cells were treated with control siRNA (siCtrl) or DDX3 siRNA (siRNA No. 3) for 24 h. Cells were infected with Yersinia WA-C(pTTSS+YopM) (middle panel) or WA-C(pTTSS) (right panel) for 90 min and cytosolic- (C) and nuclear (N) fractions were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total RSK1 present in the C- and N fraction of cells in each condition. C) Leptomycin B treatment and DDX3 knockdown increases nuclear YopM. HEK293T cells were not treated or treated with 25 nM LMB for 4 h or DDX3 siRNA (No. 3) for 24 h, infected with Yersinia WA-C(pTTSS+YopM) for 90 min and nuclear fractions of cells were investigated by anti-YopM immunoblot. Band intensities of YopM signals were determined by ImageJ. Each bar represents mean ± SEM of values from 4 experiments for LMB treatment and 3 experiments for DDX3 siRNA treatment; *p<0.05 (Bonferroni's multiple comparisons test). D) YopM_1–481 mutant defective in interaction with RSK accumulates in the nucleus and is exported from the nucleus in a LMB- and DDX3 dependent fashion. HEK293T cells were not treated (Ctrl), treated with 25 nM LMB for 4 h or DDX3 siRNA (siRNA No. 3) for 24 h and transfected with indicated expression vectors. Cytosolic- (C) and nuclear (N) fractions of cells in each condition were investigated by immunoblot using indicated antibodies. Bottom panel shows percentage of total myc-YopM present in the C- and N fraction of cells in the respective condition. See also S4 Fig for immunoprecipitaton of myc-YopM; myc-YopM_1–481 and myc-YopM_34–481 to verify the incapability of YopM lacking the C-terminus to bind RSK. E) YopM_1–481 mutant is unable to stimulate phosphorylation of RSK. Indicated myc-YopM constructs or empty vector were expressed in HEK293T cells and cytosolic (C) or nuclear (N) fractions were analyzed by Western blot using indicated antibodies.