The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor

The pathogenic yersiniae inject proteins directly into eukaryotic cells tha t interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpktA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plas ma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino termi nus, which contains the kinase domain, is sufficient to localize YpkA to th e plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-Containing stress fibers as well as block the activatio n of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl- terminal region of YpkA, which contains sequences that bear similarity to t he RhoA-binding domains of several eukaryotic RhoA-binding kinases, directl y interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA, Surprisin gly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine n ucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting tha t YpkA controls activated RhoA levels by a mechanism other than by simply b locking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interacti on with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Coll ectively, our data show that YpkA possesses both similarities and differenc es with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersin iae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.