Yersinia virulence factor YopM induces sustained RSK activation by interfering with dephosphorylation

Abstract

Background: Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells.

Methodology/principal findings: In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221.

Conclusions/significance: Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.

Figure 1. Identification of YopM interacting proteins by tandem-affinity-purification.

(A) HEK293T cells were infected with the indicated strains, lysed and extracts were subjected to western blotting with anti-YopM antibody. (B) J774A.1 cells were infected with deltaYopM(pYopM-CBP-SBP) and lysates were prepared 90 minutes after infection. After tandem affinity purification the beads were boiled and subjected to SDS-PAGE. Visible bands after Coomassie-staining were excised and subsequently analyzed by mass spectrometry.

Figure 2. Analysis of the YopM complex.

(A) Interactions were confirmed individually by GST-pull-down assays with [³⁵S]-methionine labeled in vitro translated proteins. (B) Interactions between YopM and further RSK and PKN isoforms not recovered by Tandem affinity purification, RSK3, RSK4 and PKN3, were analyzed by pull-down assays as in (A). (C) HEK293T cells were transfected with the indicated plasmids and harvested after 48 hours. Lysates were then incubated with 20 µl of Flag-sepharose and the precipitates and aliquots of the lysates before immunoprecipitation were analyzed by western blotting with the indicated antibodies.

Figure 3. YopM induces RSK activation in the absence of ERK signalling.

HEK293T were transfected with the indicated plasmids. 48 hours after transfection cells were serum-starved and 16–24 hours later were treated with 6 µM MEK-inhibitor. 60 minutes later cells were lysed and the lysates subjected to western blotting with the indicated antibodies.

Figure 4. YopM protects RSK from dephosphorylation during ATP-depletion.

48 hours after transfection of HEK293T with the indicated plasmids, the medium of the cells was exchanged with PBS supplemented with rotenone and 2-deoxyglucose. Cells were then harvested in 5 minute intervals and flash frozen. Westernblots of the lysates were then analyzed with the indicated antibodies.

Figure 5. YopM specifically protects RSK from dephosphorylation during in vitro dephosphorylation assays.

(A) 50 ng active RSK1 and 1 µg GST or 1 µg GST-YopM were preincubated in lambda-phosphatase buffer for 15 minutes on ice. Then 1 µl of lambda phosphatase was added and the reaction was transferred to 30°C under gentle constant shaking. The reaction was stopped by addition of 2x SDS buffer and boiling. After western blotting analysis was carried out with the antibodies indicated. (B) As in (A) except that RSK2, RSK3 and RSK4 were used in the reaction. (C) YopM interacts with both phosphorylated and unphosphorylated RSK1. 500 ng active RSK was pretreated for 90 minutes with lambda phosphatase, while 500 ng was left untreated. 250 ng of the dephosphorylated or the active RSK1 was then incubated with GST or GST-YopM bound to Glutathione-sepharose and processed as described under pull-down in the Experimental Procedures section. After washing the beads were boiled in 2x SDS buffer and subjected to western blotting with the antibodies stated.

Figure 6. YopM delivered during infection induces protracted activation of endogenous RSK in J774A.1 cells.

J774A.1 cell were infected with WA-C(pTTSS) (left upper panel) or with WA-C(pTTSS+pYopM) (right upper panel) or with WA-deltaYopM (left lower panel) or with WA-deltaYopM(pYopM) (right lower panel) and harvested at the indicated time points. Bacteria were killed by the addition of gentamicin after 90 minutes. Cellular lysates were then subjected to western blotting and probed with the indicated antibodies.