The Yersinia Virulence Factor YopM Forms a Novel Protein Complex with Two Cellular Kinases

Abstract

Pathogenic Yersinia contain a virulence plasmid that encodes genes for intracellular effectors, which neutralize the host immune response. One effector, YopM, is necessary for Yersinia virulence, but its function in host cells is unknown. To identify potential cellular pathways affected by YopM, proteins that co-immunoprecipitate with YopM in mammalian cells were isolated and identified by mass spectrometry. Results demonstrate that two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein kinase 1 (RSK1), interact directly with YopM. These two kinases associate only when YopM is present, and expression of YopM in cells stimulates the activity of both kinases. RSK1 is activated directly by interaction with YopM, and RSK1 kinase activity is required for YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs independently of the actions of YopJ on the MAPK pathway. YopM is also required for Yersinia-induced changes in RSK1 mobility in infected macrophage cells. These results identify the first intracellular targets of YopM and suggest YopM acts to stimulate the activity of PRK2 and RSK1.

Acetylation of MEK2 and IκB kinase (IKK) activation loop residues by YopJ inhibits signaling

“…It was verified from immunoblots by using the MEK1/2 CST9122 antibody that cotransfection of YopJ was sufficient to modify overexpressed myc-MEK2-(His) 6 in a similar fashion to endogenous MEK (data not shown). Myc-MEK2-(His) 6 was prepared by Ni-NTA affinity from lysates of HeLa cells coexpressing YopJ WT or a control plasmid. The prepared samples were analyzed for the presence of modification by Western blots by using anti-myc, anti-MEK2, and the discriminating anti-MEK1/2 CST9122 antibodies (Fig.…”

“…In the light of our results, we speculate that some of these effects might be due to the transfer of acetyl groups to the active sites of MAPKKs/IKKs catalyzed by yet uncharacterized serine/threonine acetyl transferases. pCMV-myc-MEK2-(His) 6 coding for MEK2 with a hexahistidine tag at its carboxyl terminus was constructed by amplifying the MEK2 sequence with appropriate primers and ligating into pCMV-myc (a modified pCMV5 plasmid). GST-MEK2 was made by amplifying the coding region of MEK2 and ligating in-frame with GST in the bacterial expression plasmid pGEX6P.…”

“…MEK2 bearing a myc epitope tag at its amino terminus and a hexahistidine tag at the carboxyl terminus was overexpressed in HeLa cells. It was verified from immunoblots by using the MEK1/2 CST9122 antibody that cotransfection of YopJ was sufficient to modify overexpressed myc-MEK2-(His) 6 in a similar fashion to endogenous MEK (data not shown). Myc-MEK2-(His) 6 was prepared by Ni-NTA affinity from lysates of HeLa cells coexpressing YopJ WT or a control plasmid.…”

“…Suppression of phagocytosis enables Yersinia to evade the macrophage defense network, thereby allowing them to proliferate in Peyer's patches as extracellular microcolonies. The leucine-rich protein, YopM, has been shown to bind to several host cell kinases, resulting in their activation (6). The remaining outer protein, YopJ (also called YopP in Y. enterocolitica) has emerged as an important agent that leads to the reduced host inflammatory response characteristic of Yersinia infections (5).…”

The Proteasome Pathway Destabilizes Yersinia Outer Protein E and Represses Its Antihost Cell Activities

“…In addition, YopP/YopJ triggers apoptosis in macrophages, a process that involves inhibition of the antiapoptotic NF-B pathway. Lastly, YopM forms a complex with the kinases protein kinase C-like 2 and ribosomal S6 protein kinase 1 (16). YopM is able to activate these molecules, but the precise function of YopM in the host cell is still unknown.…”