ROP18 is a polymorphic serine/threonine protein kinase of Toxoplasma gondii that is stored in the bulb of the rhoptries, the apical secretory organelles discharged at the moment of host-cell invasion. During invasion the mature kinase is released into the host cell and is tethered, through an arginine-rich amphipathic helical region upstream of its kinase domain, to the cytosolic face of the parasitophorous vacuole membrane that surrounds the intracellular parasite. From this position it phosphorylates host proteins that reach the vacuole surface. Its best characterized substrates are interferon-inducible immunity-related GTPases (IRGs) of rodents, notably Irga6, which it phosphorylates on conserved threonines in switch region I of the GTPase domain; this inactivates the GTPase, prevents IRG oligomerization and loading onto the vacuole membrane, and so protects the vacuole from being ruptured and the parasite from being destroyed in interferon-activated macrophages. ROP18 works together with the pseudokinase ROP5 and the dense granule protein GRA7, which form a vacuolar complex that presents and regulates its IRG substrates. Through its N-terminal extension ROP18 also binds the host endoplasmic reticulum-bound transcription factor ATF6beta and, in a manner requiring its catalytic activity, promotes its proteasomal degradation. The kinase is an acute virulence determinant in mice, where catalytic activity, vacuole membrane targeting and allelic form all contribute to the ability of the parasite to survive host interferon-gamma responses.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | TOO GENERAL | Summary: Generic nucleotide binding, inferred from the protein kinase ATP-binding signature. Reason: True but uninformative. ROP18 has a canonical protein kinase fold with an annotated ATP-binding residue, and the more specific ATP binding (GO:0005524) is asserted elsewhere in this review and entails nucleotide binding. Supporting Evidence: file:TOXGM/ROP18/ROP18-uniprot.txt /ligand="ATP" |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Protein kinase activity from the InterPro Ser/Thr kinase active-site signature. Reason: The claim is correct, since ROP18 is a catalytically active kinase that autophosphorylates and phosphorylates host substrates, but the specific term protein serine/threonine kinase activity (GO:0004674) is asserted in this review and entails this one. Supporting Evidence: PMID:17305424 We therefore demonstrate that rhoptries can discharge active kinases at the parasiteβhost cell interface upon cell invasion. |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Core molecular function. ROP18 is an active Ser/Thr kinase that autophosphorylates and phosphorylates host IRG GTPases on switch I threonines. Reason: Although delivered electronically, this term matches the experimentally demonstrated activity. Recombinant and parasite-derived ROP18 autophosphorylates and phosphorylates Irgb6 in vitro only when catalytically competent, and the mapped substrate sites in Irga6 are threonines in switch region I. The dual serine/threonine term is the right level of specificity for a kinase whose characterized sites are threonines but whose family and EC assignment are Ser/Thr. Supporting Evidence: PMID:21147463 When ROP18 and Irgb6 were each separately immunoprecipitated and incubated in vitro in a hot kinase reaction, both auto-phosphorylation of several forms of ROP18 and labeling of Irgb6 were readily detected by the active ROP18 but not the kinase-dead form (Figure 4B). PMID:21203588 We show here that the ROP18 kinase from highly virulent strains targets Irga6, phosphorylating two threonines in the switch I loop. |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: ATP binding by the protein kinase domain, consistent with demonstrated phosphotransfer activity. Reason: ROP18 carries an intact protein kinase domain with a conserved ATP-binding site, and catalysis has been demonstrated directly (autophosphorylation and substrate phosphorylation are lost in the kinase-dead mutant), which requires ATP binding. Supporting Evidence: file:TOXGM/ROP18/ROP18-uniprot.txt /ligand="ATP" PMID:21147463 both auto-phosphorylation of several forms of ROP18 and labeling of Irgb6 were readily detected by the active ROP18 but not the kinase-dead form (Figure 4B). |
| GO:0005634 nucleus | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter-derived nuclear localization, grafted from an animal LOK/STK10-like kinase family, and contradicted by the established cell biology of ROP18. Reason: ROP18 is a classical rhoptry bulb protein with an N-terminal signal and pro-region. It is stored in the rhoptries, discharged into the host cell at invasion, and tethered to the cytosolic face of the parasitophorous vacuole membrane, where it stays for the intracellular cycle. No study places it in the parasite or host nucleus, and the grafted PANTHER family (PTHR44167, animal ovarian-specific and LOK-related kinases) is a poor donor for an apicomplexan secreted effector whose compartments are the rhoptry and the vacuole membrane. Propagation Review Root cause: PROPAGATION BAD Sources checked: PANTHER:PTN009174714 SOURCE BAD Ancestral node of PANTHER family PTHR44167 (animal LOK/STK10-related kinases); nuclear localization of the metazoan members does not transfer to a secreted apicomplexan rhoptry kinase. Supporting Evidence: PMID:17305424 We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. PMID:22906355 Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. |
| GO:0016301 kinase activity | IEA GO_REF:0000104 | TOO GENERAL | Summary: Generic kinase activity. Reason: Correct but uninformative; entailed by protein serine/threonine kinase activity (GO:0004674), which is accepted in this review on experimental grounds. Supporting Evidence: PMID:21203588 We show here that the ROP18 kinase from highly virulent strains targets Irga6, phosphorylating two threonines in the switch I loop. |
| GO:0016740 transferase activity | IEA GO_REF:0000104 | TOO GENERAL | Summary: Root-level transferase activity. Reason: True of every kinase and entailed by protein serine/threonine kinase activity (GO:0004674); it adds nothing to the functional description of ROP18. Supporting Evidence: PMID:21203588 We show here that the ROP18 kinase from highly virulent strains targets Irga6, phosphorylating two threonines in the switch I loop. |
| GO:0044773 mitotic DNA damage checkpoint signaling | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of a metazoan cell-cycle checkpoint role onto a secreted parasite effector kinase. Reason: There is no evidence that ROP18 participates in any checkpoint. The mature protein is exported from the parasite at invasion and acts on the cytosolic face of the vacuole membrane on host substrates (IRG GTPases, and the host ER factor ATF6beta); the one replication phenotype reported for it, stimulation of intravacuolar parasite multiplication, is a downstream consequence of effector activity, not checkpoint signalling. The term is grafted from an animal kinase family (PTHR44167) and should not be propagated to an apicomplexan rhoptry kinase. Propagation Review Root cause: PROPAGATION BAD Sources checked: PANTHER:PTN009174714 SOURCE BAD Metazoan checkpoint-kinase function at the grafted PANTHER node; the apicomplexan ROP2 family members descend as secreted host-directed effectors and do not inherit this process. Supporting Evidence: PMID:22906355 Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. PMID:21670204 The kinase activity of ROP18 is essential for proteasome-dependent degradation of ATF6Ξ² and for parasite virulence. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | MODIFY | Summary: Serine-only kinase term derived from the RHEA mapping of the UniProt catalytic activity statement. Reason: The serine-specific child asserts a narrower chemistry than ROP18 displays. The experimentally mapped sites on its principal substrate Irga6 are threonines (Thr102 and Thr108) in switch region I, and the UniProt entry itself lists both the seryl and threonyl phosphotransfer reactions under EC 2.7.11.1. The dual term protein serine/threonine kinase activity (GO:0004674), already carried by this gene, is the supported level. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:21203588 We show here that the ROP18 kinase from highly virulent strains targets Irga6, phosphorylating two threonines in the switch I loop. PMID:22906355 Infection of IFN-Ξ³ activated L929 cells with wild type ROP18 led to robust phosphorylation of Thr 102 and Thr108 in Irga6, as detected using phospho-specific antibodies (Fig. 6C). |
| GO:0020008 rhoptry | IDA PMID:17305424 ROP18 is a rhoptry kinase controlling the intracellular prol... | NEW | Summary: ROP18 is stored in the bulb of the rhoptries before discharge, demonstrated by immunofluorescence and immuno-EM of tagged and endogenous protein. Reason: The rhoptry is the defining compartment of this protein family and of ROP18 specifically, yet GOA has no cellular component annotation for this entry other than the erroneous TreeGrafter nucleus term. Direct localization evidence exists in the original characterization of the protein. Supporting Evidence: PMID:17305424 We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. PMID:22906355 In these examples, ROP18 was detected within the rhoptries, but not at the PVM due to extraction with Triton X-100 used to permeabilize the cells. |
| GO:0020005 symbiont-containing vacuole membrane | IDA PMID:22906355 The arginine-rich N-terminal domain of ROP18 is necessary fo... | NEW | Summary: After rhoptry discharge the mature kinase associates with the parasitophorous vacuole membrane, on its cytosolic (host-facing) side, for the duration of the intracellular cycle. Reason: GO:0020005 is the term GO uses for the Toxoplasma parasitophorous vacuole membrane in annotations of parasite-encoded vacuolar proteins. Vacuole association is not incidental, since mutants of the arginine-rich amphipathic region retain kinase activity but, lacking PVM association, fail to phosphorylate host Irga6 and are avirulent, so the location is integral to the molecular function and belongs in the record. Supporting Evidence: PMID:22906355 Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. PMID:22906355 Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. |
| GO:0052170 symbiont-mediated suppression of host innate immune response | IMP PMID:21147463 Phosphorylation of immunity-related GTPases by a Toxoplasma ... | NEW | Summary: By phosphorylating host IRG GTPases at the vacuole membrane, ROP18 blocks their recruitment and prevents interferon-driven destruction of the parasite in activated macrophages. Reason: ROP18 itself performs the step, since it is the kinase that modifies the host effector proteins, so this is participation and not mere necessity. The genetic evidence is reciprocal, with deletion in a type I strain increasing IRG loading and clearance, and transgenic expression in an IRG-susceptible type III strain reducing IRG coating and killing. The process is recorded with the generic symbiont and host term because the IRG arm of this mechanism operates in rodents, whose expanded IRG family humans lack. Supporting Evidence: PMID:21147463 Parasites expressing active ROP18 largely resisted IRG recruitment, while efficient Irgb6 loading was observed on vacuoles containing parasites deficient in ROP18 (Figure 3A,B). PMID:21147463 Enhanced recruitment of Irgb6 to vacuoles containing Ξrop18 parasites occurred within IFN-Ξ³-activated macrophages, indicating that in the absence of ROP18, type I parasites are less able to avoid IRG recruitment (Figure 6C). PMID:22761577 Indeed, transgenic expression in the type III strain CEP of either ROP18I or ROP18II decreased the average number of vacuoles coated with Irgb6 from 45% to 23% (P = 0.001) for ROP18I or 29% (P = 0.003) for ROP18II (Figure 1B). |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Does ROP18 phosphorylate ATF6beta directly, or does it destabilize it only through N-terminal binding plus a catalysis-dependent step acting on another substrate? The published data establish kinase-dependent proteasomal degradation but not a phosphorylation site.
Suggested experts: Toxoplasma effector biologists, host ER stress and unfolded protein response researchers
Q: What does ROP18 do in human infection, where the expanded IRG family that provides its main rodent substrates is absent? If the IRG arm is rodent-specific, is the kinase retained in human isolates for the ATF6beta axis or for an unidentified human substrate?
Suggested experts: clinical toxoplasmosis researchers, comparative immunologists
Q: Is the type II (ME49) allele reviewed here functionally equivalent to the type I allele at the level of substrate phosphorylation, with virulence differences explained entirely by the ROP5 allele and expression context?
Suggested experts: Toxoplasma population genetics and virulence QTL groups
Experiment: Quantitative phosphoproteomics of host and parasite proteins in cells infected with ME49 wild-type versus a ME49 rop18 knockout and a kinase-dead complement, under naive and interferon-gamma activated conditions.
Hypothesis: Beyond Irga6 and other IRGs, ROP18 has a small, defined set of vacuole-proximal substrates whose phosphorylation depends on both catalytic activity and PVM targeting.
Type: mass spectrometry phosphoproteomics with isogenic parasite lines
Experiment: Identify the approximately 70 kDa tachyzoite protein phosphorylated by recombinant ROP18 in vitro, by substrate trapping with the kinase-dead allele followed by mass spectrometry.
Hypothesis: ROP18 has at least one intrinsic parasite substrate, which would explain the kinase-dependent effect of ROP18 overexpression on intravacuolar replication.
Type: substrate trapping and proteomics
Experiment: Express the ME49 type II allele and the type I allele in a common rop18-null background together with matched ROP5 alleles, and measure Irga6 Thr102 and Thr108 phosphorylation, IRG coating of the vacuole and survival in interferon-activated murine and human cells.
Hypothesis: Allelic differences in acute virulence are driven by the ROP5 partner rather than by intrinsic differences in ROP18 catalytic competence, and neither allele affects survival in human cells.
Type: comparative genetics with cell biological readouts
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)