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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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Demonstration of the IRAK-1/SHP-1 interaction.(A) Western blot analysis demonstrating the Co-IP of IRAK-1 and SHP-1. IRAK-1 and SHP-1 were immunoprecipitated then blotted against SHP-1 and IRAK-1 antibodies. Rabbit IgG anti-rat was used as a control. (B) In gel PTP activity assay of SHP-1 and IRAK-1 IPs (top blot). Rabbit IgG anti-rat was used as an IP control. Cell lysates of WT and SHP-1−/− were added in the last two lanes to confirm that the signal was SHP-1. Lower blot represents IRAK-1 kinase activity in reciprocal IPs. (C) In vitro transcription/translation of the IRAK-1 gene was performed using radiolabelled methionine. First lane shows the IRAK-1 input. The last two lanes show methionine-labelled IRAK-1 pulled down after 1 h incubation with either a GST-SHP-1 or GST respectively. (D) Kinase assay measuring IRAK-1 activity upon its interaction with either GST alone or increasing concentrations of an active GST-SHP-1 construct (left panel). Kinase assay measuring IRAK-4 activity (right panel) upon its interaction with either GST alone or GST-SHP-1 (5 µg). (E) Kinase assay showing IRAK-1 activity at basal level and upon LPS treatment subjected to alkali treatment to evaluate tyrosine phosphorylation in IRAK-1. Tyrosyl phosphorylation was confirmed by western blot using an anti-phosphotyrosine antibody. An IP fraction was kept and blotted for IRAK-1 as a loading control.
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pntd-0000305-g002: Demonstration of the IRAK-1/SHP-1 interaction.(A) Western blot analysis demonstrating the Co-IP of IRAK-1 and SHP-1. IRAK-1 and SHP-1 were immunoprecipitated then blotted against SHP-1 and IRAK-1 antibodies. Rabbit IgG anti-rat was used as a control. (B) In gel PTP activity assay of SHP-1 and IRAK-1 IPs (top blot). Rabbit IgG anti-rat was used as an IP control. Cell lysates of WT and SHP-1−/− were added in the last two lanes to confirm that the signal was SHP-1. Lower blot represents IRAK-1 kinase activity in reciprocal IPs. (C) In vitro transcription/translation of the IRAK-1 gene was performed using radiolabelled methionine. First lane shows the IRAK-1 input. The last two lanes show methionine-labelled IRAK-1 pulled down after 1 h incubation with either a GST-SHP-1 or GST respectively. (D) Kinase assay measuring IRAK-1 activity upon its interaction with either GST alone or increasing concentrations of an active GST-SHP-1 construct (left panel). Kinase assay measuring IRAK-4 activity (right panel) upon its interaction with either GST alone or GST-SHP-1 (5 µg). (E) Kinase assay showing IRAK-1 activity at basal level and upon LPS treatment subjected to alkali treatment to evaluate tyrosine phosphorylation in IRAK-1. Tyrosyl phosphorylation was confirmed by western blot using an anti-phosphotyrosine antibody. An IP fraction was kept and blotted for IRAK-1 as a loading control.

Mentions: Then, to evaluate whether the SHP-1 regulatory effect on IRAK-1's kinase activity involved their interaction, we performed immunoprecipitation assays and observed that IRAK-1 and SHP-1 co-IP (Figure 2A). Their association was further confirmed as we have detected PTP activity corresponding to SHP-1 in the IRAK-1 IP (Figure 2B, top panel), and IRAK-1 kinase activity in the IP of SHP-1 (Figure 2B, bottom panel). A secondary rat antibody was used as a negative control (Figures 2A and B). These experiments suggested the presence of IRAK-1 and SHP-1 in the same multi-protein complex. To test whether they directly interact, we in vitro translated IRAK-1 using radiolabelled methionine, and put the radiolabelled IRAK-1 in contact with GST-SHP-1. IRAK-1 was pulled down specifically by GST-SHP-1 and not by GST alone, showing that this interaction is direct (Figure 2C).


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)

Demonstration of the IRAK-1/SHP-1 interaction.(A) Western blot analysis demonstrating the Co-IP of IRAK-1 and SHP-1. IRAK-1 and SHP-1 were immunoprecipitated then blotted against SHP-1 and IRAK-1 antibodies. Rabbit IgG anti-rat was used as a control. (B) In gel PTP activity assay of SHP-1 and IRAK-1 IPs (top blot). Rabbit IgG anti-rat was used as an IP control. Cell lysates of WT and SHP-1−/− were added in the last two lanes to confirm that the signal was SHP-1. Lower blot represents IRAK-1 kinase activity in reciprocal IPs. (C) In vitro transcription/translation of the IRAK-1 gene was performed using radiolabelled methionine. First lane shows the IRAK-1 input. The last two lanes show methionine-labelled IRAK-1 pulled down after 1 h incubation with either a GST-SHP-1 or GST respectively. (D) Kinase assay measuring IRAK-1 activity upon its interaction with either GST alone or increasing concentrations of an active GST-SHP-1 construct (left panel). Kinase assay measuring IRAK-4 activity (right panel) upon its interaction with either GST alone or GST-SHP-1 (5 µg). (E) Kinase assay showing IRAK-1 activity at basal level and upon LPS treatment subjected to alkali treatment to evaluate tyrosine phosphorylation in IRAK-1. Tyrosyl phosphorylation was confirmed by western blot using an anti-phosphotyrosine antibody. An IP fraction was kept and blotted for IRAK-1 as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2596967&req=5

pntd-0000305-g002: Demonstration of the IRAK-1/SHP-1 interaction.(A) Western blot analysis demonstrating the Co-IP of IRAK-1 and SHP-1. IRAK-1 and SHP-1 were immunoprecipitated then blotted against SHP-1 and IRAK-1 antibodies. Rabbit IgG anti-rat was used as a control. (B) In gel PTP activity assay of SHP-1 and IRAK-1 IPs (top blot). Rabbit IgG anti-rat was used as an IP control. Cell lysates of WT and SHP-1−/− were added in the last two lanes to confirm that the signal was SHP-1. Lower blot represents IRAK-1 kinase activity in reciprocal IPs. (C) In vitro transcription/translation of the IRAK-1 gene was performed using radiolabelled methionine. First lane shows the IRAK-1 input. The last two lanes show methionine-labelled IRAK-1 pulled down after 1 h incubation with either a GST-SHP-1 or GST respectively. (D) Kinase assay measuring IRAK-1 activity upon its interaction with either GST alone or increasing concentrations of an active GST-SHP-1 construct (left panel). Kinase assay measuring IRAK-4 activity (right panel) upon its interaction with either GST alone or GST-SHP-1 (5 µg). (E) Kinase assay showing IRAK-1 activity at basal level and upon LPS treatment subjected to alkali treatment to evaluate tyrosine phosphorylation in IRAK-1. Tyrosyl phosphorylation was confirmed by western blot using an anti-phosphotyrosine antibody. An IP fraction was kept and blotted for IRAK-1 as a loading control.
Mentions: Then, to evaluate whether the SHP-1 regulatory effect on IRAK-1's kinase activity involved their interaction, we performed immunoprecipitation assays and observed that IRAK-1 and SHP-1 co-IP (Figure 2A). Their association was further confirmed as we have detected PTP activity corresponding to SHP-1 in the IRAK-1 IP (Figure 2B, top panel), and IRAK-1 kinase activity in the IP of SHP-1 (Figure 2B, bottom panel). A secondary rat antibody was used as a negative control (Figures 2A and B). These experiments suggested the presence of IRAK-1 and SHP-1 in the same multi-protein complex. To test whether they directly interact, we in vitro translated IRAK-1 using radiolabelled methionine, and put the radiolabelled IRAK-1 in contact with GST-SHP-1. IRAK-1 was pulled down specifically by GST-SHP-1 and not by GST alone, showing that this interaction is direct (Figure 2C).

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