A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.
The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.
Institute for Genetics, University of Cologne, Cologne, Germany.
Martin C
MC
Fleckenstein
Michael L
ML
Reese
Stephanie
S
Könen-Waisman
John C
JC
Boothroyd
Jonathan C
JC
Howard
Tobias
T
Steinfeldt
D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000469:parasitology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006790:Host-Parasite Interactions; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D015800:Protozoan Proteins; D037521:Virulence Factors; D013912:Threonine; D017346:Protein Serine-Threonine Kinases; C556793:ROP18 protein, Toxoplasma gondii; D020558:GTP Phosphohydrolases; C491070:Iigp1 protein, mouse