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Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif.

Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M - PLoS Negl Trop Dis (2008)

Bottom Line: We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif).This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member.We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada.

ABSTRACT
Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKalpha/beta) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

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Related in: MedlinePlus

IRAK-1/SHP-1 interaction is enhanced by Leishmania infection and leads to IRAK-1 signalling alteration and MØ functional inhibition.(A) Western blot analysis demonstrating the enhanced co-IP of IRAK-1 and SHP-1 in response to Leishmania infection. IRAK-1 was immunoprecipitated from uninfected and Leishmania-infected MØ lysates (30 min post-infection). The IPs were run on SDS-PAGE and blotted against SHP-1 (upper panel). Membrane was then stripped and blotted against IRAK-1 as a control for equal IP (lower panel). (B) A fraction of the IPs (a, upper panel) was subjected to in gel PTP assay showing higher SHP-1 activity associated with the IP of Leishmania-infected MØs over uninfected. (C) Fraction of the IP (a, upper panel) was also subjected to a phosphatase assay based on pNPP hydrolysis demonstrating a significantly higher total phosphatase activity in the IP of Leishmania-infected cells compared to uninfected controls. *, P<0.05; error bar SD. Data are the mean of four independent experiments. (D) IRAK-1 kinase assay of WT and SHP-1−/− MØs infected or not with L. donovani for 1 h (upper panel). IP fraction was kept and subjected to western blot as a loading control of IRAK-1 immunoprecipitation (lower panel). (E) NO production by Leishmania-infected WT and SHP-1−/− MØs in response to LPS. Cells have been infected with L. donovani (O/N) and stimulated with LPS for 24 h. Significant difference P<0.05, Anova test, error bar SEM. Mean of three independent experiments. (F and G) IRAK-1 Inactivation by Leishmania causes its inability to bind TRAF6. Western blot analysis showing that Leishmania causes an abrogation of the ability of IRAK-1 to bind TRAF6, but not MyD88, upon LPS stimulation. IRAK-1 was immunoprecipitated from lysates of naïve and L. donovani-infected MØ (1 h infection) stimulated or not with LPS (100 ng/ml, 1 h). The IPs were run on SDS-PAGE and blotted against MyD88 (F) and TRAF6 (G). Membranes were stripped and blotted against IRAK-1 to demonstrate equal IP.
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pntd-0000305-g007: IRAK-1/SHP-1 interaction is enhanced by Leishmania infection and leads to IRAK-1 signalling alteration and MØ functional inhibition.(A) Western blot analysis demonstrating the enhanced co-IP of IRAK-1 and SHP-1 in response to Leishmania infection. IRAK-1 was immunoprecipitated from uninfected and Leishmania-infected MØ lysates (30 min post-infection). The IPs were run on SDS-PAGE and blotted against SHP-1 (upper panel). Membrane was then stripped and blotted against IRAK-1 as a control for equal IP (lower panel). (B) A fraction of the IPs (a, upper panel) was subjected to in gel PTP assay showing higher SHP-1 activity associated with the IP of Leishmania-infected MØs over uninfected. (C) Fraction of the IP (a, upper panel) was also subjected to a phosphatase assay based on pNPP hydrolysis demonstrating a significantly higher total phosphatase activity in the IP of Leishmania-infected cells compared to uninfected controls. *, P<0.05; error bar SD. Data are the mean of four independent experiments. (D) IRAK-1 kinase assay of WT and SHP-1−/− MØs infected or not with L. donovani for 1 h (upper panel). IP fraction was kept and subjected to western blot as a loading control of IRAK-1 immunoprecipitation (lower panel). (E) NO production by Leishmania-infected WT and SHP-1−/− MØs in response to LPS. Cells have been infected with L. donovani (O/N) and stimulated with LPS for 24 h. Significant difference P<0.05, Anova test, error bar SEM. Mean of three independent experiments. (F and G) IRAK-1 Inactivation by Leishmania causes its inability to bind TRAF6. Western blot analysis showing that Leishmania causes an abrogation of the ability of IRAK-1 to bind TRAF6, but not MyD88, upon LPS stimulation. IRAK-1 was immunoprecipitated from lysates of naïve and L. donovani-infected MØ (1 h infection) stimulated or not with LPS (100 ng/ml, 1 h). The IPs were run on SDS-PAGE and blotted against MyD88 (F) and TRAF6 (G). Membranes were stripped and blotted against IRAK-1 to demonstrate equal IP.

Mentions: Having previously reported that Leishmania can rapidly induce host PTP SHP-1 to inactivate JAK and MAP kinase pathways [8],[20], we hypothesized that the Leishmania-induced IRAK-1 inactivation observed was associated with an increased SHP-1/IRAK-1 interaction. We indeed noticed by Western blot that a significantly greater amount of SHP-1 was co-immunoprecipitated with IRAK-1 upon Leishmania infection (Figure 7A). Similarly, using in gel PTP assay, we were able to detect more SHP-1 activity in IRAK-1 IP from lysates of Leishmania-infected MØs (Figure 7B). Higher SHP-1 activity in Leishmania infected MØs was further supported when equal IP fractions were subjected to a pNPP phosphatase assay (Figure 7C).


Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionarily conserved KTIM motif.

Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B, Olivier M - PLoS Negl Trop Dis (2008)

IRAK-1/SHP-1 interaction is enhanced by Leishmania infection and leads to IRAK-1 signalling alteration and MØ functional inhibition.(A) Western blot analysis demonstrating the enhanced co-IP of IRAK-1 and SHP-1 in response to Leishmania infection. IRAK-1 was immunoprecipitated from uninfected and Leishmania-infected MØ lysates (30 min post-infection). The IPs were run on SDS-PAGE and blotted against SHP-1 (upper panel). Membrane was then stripped and blotted against IRAK-1 as a control for equal IP (lower panel). (B) A fraction of the IPs (a, upper panel) was subjected to in gel PTP assay showing higher SHP-1 activity associated with the IP of Leishmania-infected MØs over uninfected. (C) Fraction of the IP (a, upper panel) was also subjected to a phosphatase assay based on pNPP hydrolysis demonstrating a significantly higher total phosphatase activity in the IP of Leishmania-infected cells compared to uninfected controls. *, P<0.05; error bar SD. Data are the mean of four independent experiments. (D) IRAK-1 kinase assay of WT and SHP-1−/− MØs infected or not with L. donovani for 1 h (upper panel). IP fraction was kept and subjected to western blot as a loading control of IRAK-1 immunoprecipitation (lower panel). (E) NO production by Leishmania-infected WT and SHP-1−/− MØs in response to LPS. Cells have been infected with L. donovani (O/N) and stimulated with LPS for 24 h. Significant difference P<0.05, Anova test, error bar SEM. Mean of three independent experiments. (F and G) IRAK-1 Inactivation by Leishmania causes its inability to bind TRAF6. Western blot analysis showing that Leishmania causes an abrogation of the ability of IRAK-1 to bind TRAF6, but not MyD88, upon LPS stimulation. IRAK-1 was immunoprecipitated from lysates of naïve and L. donovani-infected MØ (1 h infection) stimulated or not with LPS (100 ng/ml, 1 h). The IPs were run on SDS-PAGE and blotted against MyD88 (F) and TRAF6 (G). Membranes were stripped and blotted against IRAK-1 to demonstrate equal IP.
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pntd-0000305-g007: IRAK-1/SHP-1 interaction is enhanced by Leishmania infection and leads to IRAK-1 signalling alteration and MØ functional inhibition.(A) Western blot analysis demonstrating the enhanced co-IP of IRAK-1 and SHP-1 in response to Leishmania infection. IRAK-1 was immunoprecipitated from uninfected and Leishmania-infected MØ lysates (30 min post-infection). The IPs were run on SDS-PAGE and blotted against SHP-1 (upper panel). Membrane was then stripped and blotted against IRAK-1 as a control for equal IP (lower panel). (B) A fraction of the IPs (a, upper panel) was subjected to in gel PTP assay showing higher SHP-1 activity associated with the IP of Leishmania-infected MØs over uninfected. (C) Fraction of the IP (a, upper panel) was also subjected to a phosphatase assay based on pNPP hydrolysis demonstrating a significantly higher total phosphatase activity in the IP of Leishmania-infected cells compared to uninfected controls. *, P<0.05; error bar SD. Data are the mean of four independent experiments. (D) IRAK-1 kinase assay of WT and SHP-1−/− MØs infected or not with L. donovani for 1 h (upper panel). IP fraction was kept and subjected to western blot as a loading control of IRAK-1 immunoprecipitation (lower panel). (E) NO production by Leishmania-infected WT and SHP-1−/− MØs in response to LPS. Cells have been infected with L. donovani (O/N) and stimulated with LPS for 24 h. Significant difference P<0.05, Anova test, error bar SEM. Mean of three independent experiments. (F and G) IRAK-1 Inactivation by Leishmania causes its inability to bind TRAF6. Western blot analysis showing that Leishmania causes an abrogation of the ability of IRAK-1 to bind TRAF6, but not MyD88, upon LPS stimulation. IRAK-1 was immunoprecipitated from lysates of naïve and L. donovani-infected MØ (1 h infection) stimulated or not with LPS (100 ng/ml, 1 h). The IPs were run on SDS-PAGE and blotted against MyD88 (F) and TRAF6 (G). Membranes were stripped and blotted against IRAK-1 to demonstrate equal IP.
Mentions: Having previously reported that Leishmania can rapidly induce host PTP SHP-1 to inactivate JAK and MAP kinase pathways [8],[20], we hypothesized that the Leishmania-induced IRAK-1 inactivation observed was associated with an increased SHP-1/IRAK-1 interaction. We indeed noticed by Western blot that a significantly greater amount of SHP-1 was co-immunoprecipitated with IRAK-1 upon Leishmania infection (Figure 7A). Similarly, using in gel PTP assay, we were able to detect more SHP-1 activity in IRAK-1 IP from lysates of Leishmania-infected MØs (Figure 7B). Higher SHP-1 activity in Leishmania infected MØs was further supported when equal IP fractions were subjected to a pNPP phosphatase assay (Figure 7C).

Bottom Line: We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif).This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member.We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada.

ABSTRACT
Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKalpha/beta) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.

Show MeSH
Related in: MedlinePlus