Abstract
The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the 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 activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly 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. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.
Highlights
Several Gram-negative bacteria species that live in close association, for at least part of their life cycle, with eukaryotic cells possess a “protein injection system” (designated as type III (Ref. 1)) that delivers bacterially encoded proteins directly into eukaryotic cells following attachment of the bacterium to the host cell [2]
The morphology of HeLa cells infected with Yersinia expressing the D270A variant of Yersinia protein kinase A (YpkA) was indistinguishable from that of HeLa cells infected with wild type YpkA-expressing Yersinia (Fig. 1)
1 h after the onset of infection, slightly fewer actin-containing stress fibers were observed in HeLa cells infected with Yersinia expressing full-length YpkA compared with cells infected with Yersinia expressing the YpkA⌬543–640 variant
Summary
We were interested in identifying the domains of YpkA that are required for its morphological altering properties and its intracellular targeting to the plasma membrane. We found that YpkA shares both similarities and differences with the eukaryotic RhoA-binding kinases perhaps indicating that ypkAs ancestral gene, which was likely “captured” from a eukaryotic organism by the yersiniae at some point in the past, underwent extensive modification in order to serve the needs of a prokaryotic pathogen
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