The capacity of these three pathogenic Yersinia strains to multiply extracellularly and inhibit internalization by host cells depends on a virulence plasmid that encodes a common type three secretion Folinic acid calcium salt pentahydrate system and virulence effectors such as the Yersinia outer proteins. The Yops include YopH, YopE, YopJ, YopM, YpkA, and YopK. Upon intimate contact with a target cell, these effectors are induced in the bacterium and delivered into the interacting host cell via a mechanism involving the plasmid encoded T3SS. Inside the target cell, the Yop effectors interfere with several key mechanisms of the host immune defense including phagocytosis, production of pro-inflammatory signaling molecules, and activation of the adaptive immune system. Intracellular growth is however not dependent on the virulence plasmid. Although most of the Yop effectors are necessary for Yersinia virulence, the exact mechanism underlying their individual roles is known only for a few. For example, YopE is a Rho GAP protein that mediates effects on the actin cytoskeleton and YopH is a tyrosine phosphatase that disrupts host cell signaling necessary for phagocytosis. This favours antiphagocytosis allowing bacteria to preferentially replicate extracellularly. YopD, along with YopB and LcrV, is required for translocation of Yop effectors across the host cell plasma membrane. YopB and YopD contain hydrophobic domains indicative of transmembrane proteins and constituents of a pore. It is assumed that the Yop effectors pass through this pore when Gentamycin Sulfate crossing the eukaryotic target cell membrane. Interestingly, yopK mutants form a larger pore and in line with this notion, yopK mutants overtranslocate Yop effectors. The aim of the present study was to elucidate a possible YopK effector function inside the host cell. We identified the eukaryotic signaling protein called receptor for activated C kinase as a potential target of YopK. RACK1 is a cytosolic WD-40 repeat protein that was originally identified as being bound to and stabilizing the active form of protein kinase C. Additionally, RACK1 binds to b1-integrins, which also function as Yersinia receptors on the target cell surface. We found that YopK is required for productive delivery of Yop effectors and, together with an interaction with RACK1, is crucial for Yersinia antiphagocytosis. RACK1 is a ubiquitously expressed protein that interacts with a large array of signaling molecules, regulating cellular functions such as adhesion, movement, and division. In particular, RACK1 interacts with the cytoplasmic domain of b1-integrins and was also recently reported to bind focal adhesion kinase and participate in signaling from adhesion receptors. Given that b1-integrins constitute the eukaryotic cell receptors to which Yersinia pseudotuberculosis docks via its adhesin invasion, perhaps RACK1 participates in the bacterium-induced b1-integrin-mediated events. This idea was appealing because it could also mean that YopK binds to RACK1 to directly interfere with a signaling pathway that is important for host cell defense against the pathogen. To test this idea, we exposed HeLa cells to lentivirusmediated RNAi of RACK1 to generate stable cell lines with downregulated RACK1 expression. A stable clone with 85% reduction of RACK1 expression was selected for further investigation. Inhibition of internalization of the bacteria by eukaryotic cells has been demonstrated using professional phagocytes and was initially designated antiphagocytosis. Since the underlying mechanism of inhibition of internalization is similar in phagocytes and HeLa cells, the term antiphagocytosis is hereafter used for defining inhibition of bacterial internalization by both cell types. Non-opsonised Y. pseudotuberculosis is mainly internalized via the invasin-b1-integrin interaction in both these cell types. To ascertain whether RACK1 is involved in phagocytosis of Y. pseudotuberculosis or if it interferes with antiphagocytosis, we performed infection experiments using RACK1 RNAi cells and determined the amount of extracellular and intracellular bacteria.