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. 2010 Sep 10;285(37):28731-40.
doi: 10.1074/jbc.M110.112359. Epub 2010 Jul 12.

Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6

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Free PMC article

Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6

Yi-Ching Ong et al. J Biol Chem. .
Free PMC article

Abstract

The obligate intracellular parasite, Toxoplasma gondii, modulates host immunity in a variety of highly specific ways. Previous work revealed a polymorphic, injected parasite factor, ROP16, to be a key virulence determinant and regulator of host cell transcription. These properties were shown to be partially mediated by dysregulation of the host transcription factors STAT3 and STAT6, but the molecular mechanisms underlying this phenotype were unclear. Here, we use a Type I Toxoplasma strain deficient in ROP16 to show that ROP16 induces not only sustained activation but also an extremely rapid (within 1 min) initial activation of STAT6. Using recombinant wild-type and kinase-deficient ROP16, we demonstrate in vitro that ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation, Tyr(641). Furthermore, ROP16 co-immunoprecipitates with STAT6 from infected cells. Taken together, these data strongly suggest that STAT6 is a direct substrate for ROP16 in vivo.

Figures

FIGURE 1.
FIGURE 1.
ROP16 is an active kinase. In vitro kinase assays were performed using WT or kinase-deficient (KD) K404N point mutants of ROP16-HA, obtained either by immunoprecipitation from Type I parasites ectopically expressing these proteins (A) or by recombinant expression in E. coli and purification (B). Kinase reactions including [γ-32P]ATP were performed directly on the immunocomplexes (A) or with the recombinant proteins (B) for 30 min at 30 °C. Following SDS-PAGE and transfer to PVDF membranes, 32P incorporation was measured by PhosphorImager, and membranes were immunoblotted (IB) for ROP16-HA. Molecular masses are noted in kDa on the left.
FIGURE 2.
FIGURE 2.
Deletion of the ROP16 locus identifies STAT6 as a potential target. A, schematic of Δrop16 generation (not to scale). The top line shows the construct used with the Toxoplasma sequences derived from the ROP16 flanks shown as a thick line. Bent arrows indicate promoters driving the negative (GFP) and positive (HXGPRT) selectable markers. The middle line represents the region of chromosome VIIb containing ROP16. The bottom line shows the resulting deletion of the ROP16 locus. Primers used for PCR detection of homologous integration of the targeting construct are indicated by horizontal arrows. B, confirmation of replacement of the ROP16 locus with HXGPRT by PCR. Correct 5′- and 3′-integration was demonstrated with primer pairs 1/2 and 3/4 of Fig. 2A, respectively. Primers 5 and 6 were used to demonstrate the loss of the ROP16 coding region in the Δrop16 mutant. Toxoplasma surface antigen 1 (SAG1) primers were used as a positive control for the PCRs. C and D, microarray analysis of HFFs infected with either WT or Δrop16 (KO) parasites at 1 h (C) and 5 h (D) postinfection. Expression data for HFFs 5 h post-infection with ROP16-complemented Δrop16 parasites or control Δrop16 parasites that underwent transfection and selection alongside the complemented parasites (KO.c) are shown in D. Two biological replicates of each infection per time point were analyzed. Genes shown are those identified as significantly up-regulated by SAM analysis in cells infected with WT versus Δrop16 parasites and >2-fold change in expression (C) or >3-fold change in expression in cells infected with either WT or complemented parasites versus Δrop16 parasites (D). Genes that have previously been characterized as STAT6-regulated are highlighted in gray; genes not known to be regulated by STATs are in white.
FIGURE 3.
FIGURE 3.
STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A, parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B, HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.
FIGURE 4.
FIGURE 4.
ROP16 has tyrosine kinase activity. A, Antibody Beacon tyrosine kinase detection complex was incubated with either equivalent amounts of recombinant ROP16-WT (rWT) and kinase-deficient ROP16-K404N (rKD) or 0.6 μg of JAK3 (rJAK3) as described under “Experimental Procedures”; tyrosine kinase peptide substrate (poly(Glu-Tyr), 4:1) and ATP were then added to each well, and the reactions were incubated at 30 °C for 30 min. Fluorescence was measured in a fluorescence microplate reader using excitation at 492 nm and emission at 517 nm. Background fluorescence, or fluorescence from reaction mixtures lacking kinase, was subtracted to normalize samples. Each sample was analyzed in triplicate; data shown are from one assay representative of at least three independent replicates. B, phosphoamino acid analysis of 32P-labeled ROP16 by semidry cellulose thin layer electrophoresis. Left, photograph of the TLC plate stained with ninhydrin; positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards are circled and labeled. Right, autoradiograph of the TLC plate.
FIGURE 5.
FIGURE 5.
A pan-JAK inhibitor inhibits ROP16 in vitro. ROP16 tyrosine kinase activity was assessed by fluorescence essentially as described in the legend to Fig. 4. Recombinant ROP16-WT (rWT) or recombinant JAK3 (rJAK3) were incubated with detection complex and either DMSO or pan-JAK inhibitor at the indicated concentrations for 15 min at room temperature; ATP was then added to each well, and reactions were incubated at 30 °C for 30 min. Fluorescence (excitation at 492 nm and emission at 517 nm) was measured at 1 and 30 min post-ATP addition. Background fluorescence at 1 min was subtracted; changes in fluorescence for recombinant ROP16-WT (black bars) and recombinant JAK3 (gray bars) reactions are shown normalized to DMSO-treated reactions.
FIGURE 6.
FIGURE 6.
Tyrosine 641 of STAT6 is directly phosphorylated by ROP16. Recombinant STAT6 (rSTAT6) was incubated with recombinant JAK3 (rJAK3) or recombinant (E. coli-produced) ROP16-HA (WT) or the kinase-deficient K404N (KD) in kinase assay conditions for 30 min at 30 °C, followed by SDS-PAGE and immunoblotting (IB) using antibodies specific for STAT6 phosphorylated on the key activation residue for STAT6, tyrosine 641 (Y641; top), antibodies that recognize STAT6 regardless of activation state (middle), or antibodies for HA-tagged ROP16 (bottom). Molecular masses are noted in kDa on the left.
FIGURE 7.
FIGURE 7.
Co-immunoprecipitation of ROP16 and STAT6 in infected cells. HFFs were infected at MOI ∼10 with Type I parasites ectopically expressing HA-tagged wild type ROP16 (WT) or ROP16-K404N (KD). 2 h postinfection, cells were washed with cold PBS and lysed in situ. Immunoprecipitation was carried out on clarified lysate (input) for 12 h at 4 °C using either nonspecific rabbit IgG-conjugated to agarose beads (Control IP) or anti-STAT6 polyclonal rabbit IgG conjugated to agarose beads (STAT6 IP). Immunocomplexes were washed in lysis buffer at 4 °C and then eluted in SDS sample buffer. Input and eluates were analyzed by SDS-PAGE and immunoblotting (IB) using antibodies specific for STAT6 (top panel), HA epitope of ROP16 (second panel), and parasite-secreted proteins ROP1 (third panel) and toxofilin (bottom panel). Cell equivalents are noted (1× or 20×) at the top; molecular masses are noted in kDa on the left.

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