title: The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor.
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.
authors.affiliation: Department of Cell & Molecular Biology, Immunology Section, Lund University, SE-223 62 Lund, Sweden.
authors.forename: J M
authors.initials: JM
authors.lastname: Dukuzumuremyi
authors.forename: R
authors.initials: R
authors.lastname: Rosqvist
authors.forename: B
authors.initials: B
authors.lastname: Hallberg
authors.forename: B
authors.initials: B
authors.lastname: Akerström
authors.forename: H
authors.initials: H
authors.lastname: Wolf-Watz
authors.forename: K
authors.initials: K
authors.lastname: Schesser
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000502:physiology*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D022502:Stress Fibers / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014774:Virulence; D015007:Yersinia / Q000201:enzymology* / Q000378:metabolism / Q000472:pathogenicity*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein