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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

Mutation of IRAK-1's KTIM abrogates its ability to bind SHP-1.Top panel represents methionine-labelled WT-IRAK-1 as well as the different IRAK-1 mutants in vitro translated and put in contact with 5 µg of either GST alone or a trapping GST-SHP-1 construct. Bottom panel represents equal fractions of the in vitro translated products ran on an SDS gel to show equal input. All results are representative of at least three independent experiments.
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pntd-0000305-g003: Mutation of IRAK-1's KTIM abrogates its ability to bind SHP-1.Top panel represents methionine-labelled WT-IRAK-1 as well as the different IRAK-1 mutants in vitro translated and put in contact with 5 µg of either GST alone or a trapping GST-SHP-1 construct. Bottom panel represents equal fractions of the in vitro translated products ran on an SDS gel to show equal input. All results are representative of at least three independent experiments.

Mentions: At the view of our observations, we screened the mouse IRAK-1 sequence for possible SHP-1 binding sites. We discovered that IRAK-1 contains an ITIM-like motif (286LVYGFL291) located in its kinase domain (Figure S1). This motif was found to be absent in all the other IRAK family members since the last residue is a methionine instead of a leucine (Figure S2). To determine the involvement of this ITIM-like motif in the SHP-1/IRAK-1 binding, we used the full-length IRAK-1 sequence to introduce site-specific mutations within the motif followed by in vitro binding assays (Figure 3). Firstly, a Y288F mutation slightly decreased SHP-1 binding suggesting that possible phosphorylation of the motif's central tyrosine may increase binding affinity but is not absolutely necessary for the binding to occur. Secondly, an L291M mutation, which renders the site no more ITIM-like, significantly decreased SHP-1 binding. Thirdly, the G289A/F290Y/L291M triple mutation, which also disrupts the ITIM-like motif, completely abrogated the binding of SHP-1. Interestingly, this triple mutant of IRAK-1 is identical to the corresponding site within IRAK-4. Collectively, these site-specific mutations confirm the role of the ITIM-like motif in the binding of SHP-1 to IRAK-1. This represents the first description of such a motif in a kinase that we now call KTIM (Kinase Tyrosyl-based Inhibitory Motif). Importantly, these experiments also suggest that the SHP-1-mediated regulation of IRAK-1 is a mechanism not shared with IRAK-4.


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)

Mutation of IRAK-1's KTIM abrogates its ability to bind SHP-1.Top panel represents methionine-labelled WT-IRAK-1 as well as the different IRAK-1 mutants in vitro translated and put in contact with 5 µg of either GST alone or a trapping GST-SHP-1 construct. Bottom panel represents equal fractions of the in vitro translated products ran on an SDS gel to show equal input. All results are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000305-g003: Mutation of IRAK-1's KTIM abrogates its ability to bind SHP-1.Top panel represents methionine-labelled WT-IRAK-1 as well as the different IRAK-1 mutants in vitro translated and put in contact with 5 µg of either GST alone or a trapping GST-SHP-1 construct. Bottom panel represents equal fractions of the in vitro translated products ran on an SDS gel to show equal input. All results are representative of at least three independent experiments.
Mentions: At the view of our observations, we screened the mouse IRAK-1 sequence for possible SHP-1 binding sites. We discovered that IRAK-1 contains an ITIM-like motif (286LVYGFL291) located in its kinase domain (Figure S1). This motif was found to be absent in all the other IRAK family members since the last residue is a methionine instead of a leucine (Figure S2). To determine the involvement of this ITIM-like motif in the SHP-1/IRAK-1 binding, we used the full-length IRAK-1 sequence to introduce site-specific mutations within the motif followed by in vitro binding assays (Figure 3). Firstly, a Y288F mutation slightly decreased SHP-1 binding suggesting that possible phosphorylation of the motif's central tyrosine may increase binding affinity but is not absolutely necessary for the binding to occur. Secondly, an L291M mutation, which renders the site no more ITIM-like, significantly decreased SHP-1 binding. Thirdly, the G289A/F290Y/L291M triple mutation, which also disrupts the ITIM-like motif, completely abrogated the binding of SHP-1. Interestingly, this triple mutant of IRAK-1 is identical to the corresponding site within IRAK-4. Collectively, these site-specific mutations confirm the role of the ITIM-like motif in the binding of SHP-1 to IRAK-1. This represents the first description of such a motif in a kinase that we now call KTIM (Kinase Tyrosyl-based Inhibitory Motif). Importantly, these experiments also suggest that the SHP-1-mediated regulation of IRAK-1 is a mechanism not shared with IRAK-4.

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