The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs

  • Quentin Deveuve
  • Kevin Lesage
  • Thomas Mouveaux
  • Mathieu Gissot
Original Paper

Abstract

Toxoplasma gondii has a complex life cycle characterized by multiple differentiation steps that are essential for its survival in both human and definitive feline host. Several studies have demonstrated the importance of phosphorylations by protein kinases during the life cycle of T. gondii. However, very little is known about protein phosphatases and their regulators in the parasite. We report the molecular and functional characterization of the T. gondii ortholog of the inhibitor-2 protein, designated TgI2. We show that TgI2 encompasses conserved motifs involved in the interaction and modulation of the phosphatase activity of T. gondii protein phosphatase 1, named TgPP1. We show that a specific combination of motifs is involved in binding and/or inhibition of the TgPP1 activity. We show here that the TgI2 protein is a potent inhibitor of TgPP1 phosphatase activity. TgI2 SILK and RVxF motifs are critical for regulating the activity of TgPP1, a feature that is common with the higher eukaryotes inhibitor-2 protein.

Keywords

Toxoplasma Phosphatase PP1 Inhibitor-2 Plasmodium Cell-cycle SILK HYNE RVxF 

Supplementary material

436_2017_5543_MOESM1_ESM.pdf (765 kb)
Figure S1Purification of TgI2-GST and TgPP1-His tagged proteins. A: A SDS-PAGE gel stained with Coomassie-blue representing the total protein extracts before induction (NI, non-induced) and after induction (I, induced) of the expression of the TgI2-GST protein. The purified TgI2-GST protein is also represented (E, elution). Molecular weight marker (M) is on the left side of the Fig. B: A SDS-PAGE gel stained with Coomassie-blue representing the total protein extracts before induction (NI, non-induced) and after induction (I, induced) of the expression of the TgPP1-His protein. The purified TgPP1-His protein is also represented (E, elution). Molecular weight marker (M) is on the left side of the Fig. C: Graphical representation of the sequences mutated on the TgI2-GST protein. The name of the putative TgPP1-binding motif is indicated on the top of the figure. The sequence of the wild-type motif is indicated in black and the number corresponds to the aa position in the protein. The mutated aa are indicated in red. D: A SDS-PAGE gel stained with Coomassie-blue representing the purified mutated TgI2-GST proteins. The purified TgI2 proteins mutated for the SILK domain (lane 1), the RVxF domain (Lane 2), the HYNE domain (lane 3), the SILK and RVxF domains (lane 4), the SILK and HYNE domain (lane 5), the RvxF and HYNE domains (lane 6) and the SILK, RVxF and HYNE domains (lane 7) are represented. Molecular weight marker (M) is on the left side of the figure. (PDF 765 kb)
436_2017_5543_MOESM2_ESM.pdf (95 kb)
Figure S2GST is not able to inhibit TgPP1 activity. Graphical representation of the TgPP1 activity after incubation with different concentrations of the GST protein (indicated at the bottom of the figure). Activity is represented as a % of the phosphatase activity measured without inhibitor. Experiment was repeated three times with duplicate measurement. ns: non-significant. (PDF 95 kb)
436_2017_5543_MOESM3_ESM.pdf (68 kb)
Figure S3TgI2 and TgMIC1 colocalization. IFA analysis of the TgI2-HA (green) and TgMIC1 (red) localization in the tachyzoite. DAPI (blue) was used to label DNA. The phase-contrast is also presented. The scale bar is indicated at the bottom right of each picture. (PDF 68 kb)
436_2017_5543_MOESM4_ESM.docx (15 kb)
Table S1(DOCX 15 kb)

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d’Infection et d’Immunité de LilleLilleFrance

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