A Toxoplasma gondii-derived ROP38 protein engages Ser15/Thr18-linked p53 activation and DNA damage-associated apoptotic signaling in human cancer cells.

Abstract

Background: The tumor suppressor p53 integrates diverse cellular stresses to regulate DNA damage responses, cell-cycle progression, and apoptosis-associated signaling. Toxoplasma gondii secretes rhoptry effectors that remodel host signaling networks, but the host targets and signaling functions of the rhoptry kinase ROP38 remain poorly defined.

Methods: GFP-tagged T. gondii ME49 ROP38 full-length protein (ROP38 Full) and its kinase-domain fragment (ROP38 KiD) were stably expressed in HCT116 and HepG2 human cancer cells. Kinase-associated activity was assessed in anti-GFP immunoprecipitates, and a candidate co-immunoprecipitation screen was used to identify ROP38-associated host proteins. ROP38-p53 association and phospho-p53 species were further examined by co-immunoprecipitation. Purified recombinant ROP38 and human p53 were used for direct interaction and in vitro phosphorylation assays. Whole-cell p53 signaling, DNA damage/apoptosis-associated markers, and cell-cycle distribution were also analyzed. Statistical analyses used one- or two-way ANOVA, as appropriate, followed by Tukey's multiple comparisons test.

Results: ROP38 Full- and ROP38 KiD-containing immunoprecipitates showed increased kinase-associated activity. Among the candidate proteins examined, p53 was the only ROP38-associated host factor detected in both cell lines. ROP38 Full and ROP38 KiD co-immunoprecipitated with total p53 and preferentially enriched p53 phosphorylated at Ser15 and Thr18. Purified recombinant ROP38 directly associated with recombinant p53 and promoted Ser15/Thr18 phosphorylation in the presence of ATP. At the whole-cell level, ROP38 expression increased p53 abundance and multisite phosphorylation, with prominent Ser15/Thr18 induction, and was accompanied by increased γH2AX, PUMA, cleaved caspase-3, and p21 expression. ROP38-expressing cells also showed decreased G₁-phase populations with increased S- and G₂/M-phase populations.

Conclusions: These data identify a candidate ROP38-associated p53 signaling module in human cancer cells and provide biochemical evidence that ROP38 can directly associate with p53 and promote Ser15/Thr18 phosphorylation in vitro. The requirement for intrinsic ROP38 catalytic activity and the physiological relevance of this relationship during T. gondii infection remain to be established.