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Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Tun-Kyi A, Finn G, Greenwood A, Nowak M, Lee TH, Asara JM, Tsokos GC, Fitzgerald K, Israel E, Li X, Exley M, Nicholson LK, Lu KP - Nat. Immunol. (2011)

Bottom Line: Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms.The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown.We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

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Pin1 is essential for IRAK1 activation upon TLR ligation(a) Pin1 KO completely blocks IRAK1 activation in mouse cells following TLR7 stimulation. Pin1 WT and KO Flt3-derived pDCs (bottom) or TLR7-expressing MEF cells (top) were simulated with R-848 for the indicated times and analyzed for the characteristic IRAK1 shift by immunoblotting with IRAK1 antibodies, with IRAK4 and Pin1 amounts as controls.(b) Pin1 KO completely blocks activation of IRAK1, but not IRAK4 following TLR7 stimulation. Peritoneal macrophages from Pin1 WT and KO mice were stimulated with R-848 for the indicated times and kinase activity of IRAK1 and IRAK4 was assessed by an IP kinase autophosphorylation assay. IRAK1, IRAK4 and Pin1 protein were assayed as controls. (c) Pin1 knockdown blocks IRAK1 activation in human cells following TLR7 and TLR9, but not TLR3 stimulation. Human THP1 monocytes were infected with viral control shRNA or shRNA targeting Pin1 and simulated with poly (I:C) (TLR3), R-848 or CpG ligands for the indicated times, followed by analyzing the characteristic IRAK1 shift using immunoblotting.(d) In vivo kinase assay demonstrates IRAK1 kinase activity in Pin1 WT, but not Pin1 KO cells. Retroviral FLAG-IRAK1, and KD-IRAK1 or vector (VCT) control were coexpressed with a HA-N-terminal 220 aa fragment of IRAK1 as a substrate in Pin1 WT and KO MEFs (schematic diagram). IRAK1 kinase activity was determined by immunoblotting with HA antibodies to assess the characteristic mobility shift in IRAK1 N-terminal 220 aa due to trans-phosphorylation by co-expressed IRAK1 proteins.(e) Pin1 KO abolishes TLR dependent activation of exogenous IRAK1 in vivo. FLAG-IRAK1 and its KD mutant were co-expressed with TLR7 in Pin1 WT and KO MEF cells using retroviral vectors and stimulated with R-848 for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting.(f) Pin1, but not its WW domain-binding mutant (W34A) or catalytically inactive PPIase domain mutant (K63A), fully rescues IRAK1 activation in Pin1 KO cells. Pin1 KO MEFs stably expressing FLAG-IRAK1 were transfected with either WT-Pin1, K63A-Pin1, W34A-Pin1 or PPIase domain of Pin1 and TLR7 and stimulated for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. Results are representative of at least three independent experiments.
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Figure 4: Pin1 is essential for IRAK1 activation upon TLR ligation(a) Pin1 KO completely blocks IRAK1 activation in mouse cells following TLR7 stimulation. Pin1 WT and KO Flt3-derived pDCs (bottom) or TLR7-expressing MEF cells (top) were simulated with R-848 for the indicated times and analyzed for the characteristic IRAK1 shift by immunoblotting with IRAK1 antibodies, with IRAK4 and Pin1 amounts as controls.(b) Pin1 KO completely blocks activation of IRAK1, but not IRAK4 following TLR7 stimulation. Peritoneal macrophages from Pin1 WT and KO mice were stimulated with R-848 for the indicated times and kinase activity of IRAK1 and IRAK4 was assessed by an IP kinase autophosphorylation assay. IRAK1, IRAK4 and Pin1 protein were assayed as controls. (c) Pin1 knockdown blocks IRAK1 activation in human cells following TLR7 and TLR9, but not TLR3 stimulation. Human THP1 monocytes were infected with viral control shRNA or shRNA targeting Pin1 and simulated with poly (I:C) (TLR3), R-848 or CpG ligands for the indicated times, followed by analyzing the characteristic IRAK1 shift using immunoblotting.(d) In vivo kinase assay demonstrates IRAK1 kinase activity in Pin1 WT, but not Pin1 KO cells. Retroviral FLAG-IRAK1, and KD-IRAK1 or vector (VCT) control were coexpressed with a HA-N-terminal 220 aa fragment of IRAK1 as a substrate in Pin1 WT and KO MEFs (schematic diagram). IRAK1 kinase activity was determined by immunoblotting with HA antibodies to assess the characteristic mobility shift in IRAK1 N-terminal 220 aa due to trans-phosphorylation by co-expressed IRAK1 proteins.(e) Pin1 KO abolishes TLR dependent activation of exogenous IRAK1 in vivo. FLAG-IRAK1 and its KD mutant were co-expressed with TLR7 in Pin1 WT and KO MEF cells using retroviral vectors and stimulated with R-848 for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting.(f) Pin1, but not its WW domain-binding mutant (W34A) or catalytically inactive PPIase domain mutant (K63A), fully rescues IRAK1 activation in Pin1 KO cells. Pin1 KO MEFs stably expressing FLAG-IRAK1 were transfected with either WT-Pin1, K63A-Pin1, W34A-Pin1 or PPIase domain of Pin1 and TLR7 and stimulated for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. Results are representative of at least three independent experiments.

Mentions: Given that Pin1 binds to and isomerizes multiple pSer-Pro motifs in IRAK1 upon TLR activation, a key question is whether Pin1 regulates IRAK1 function in TLR signaling. Therefore, we examined the effects of Pin1 KO on IRAK1 activation in response to activation of various TLRs using Pin1 WT and KO MEFs and pDCs. Although TLR7 and TLR9 ligation activated IRAK1 in a time-dependent fashion in both Pin1 WT cells (Fig. 4a), as indicated by the mobility shift and increased kinase activity (Fig 4b), as previously described12, 13, 17, there was no evidence for IRAK1 activation in either assay in Pin1 KO MEFs or pDCs (Fig. 4a, b) or in Pin1-silenced THP1 cells using RNAi (Fig. 4c). Moreover, Pin1 KO also completely abolished IRAK1 activation in response to ligation of other TLRs including TLR2 and TLR4 (Supplementary Fig. 7a, b). These effects were highly specific because Pin1 KO did not affect activation of the IRAK1 upstream kinase IRAK4 (Fig. 4b), or mitogen-activated protein kinases (MAPK) including ERKs, JNKs and p38 MAPKs upon TLR activation (Supplementary Fig. 8). Similar observations were made following LPS stimulation of macrophages (Supplementary Fig. 9a). We also assessed the effects of Pin1 deficiency on IκB degradation following pDC stimulation with R-848 and CpG or treatment of macrophages with LPS and did not see any obvious difference between Pin1 WT and KO cells (Supplementary Fig. 9b, c). To further confirm this effect of Pin1 on IRAK1 activation, we developed an assay to measure the kinase activity of IRAK1 in cells utilizing the fact that IRAK1 can phosphorylate the N-terminal 220 amino acid IRAK1 fragment containing the UD in trans, as shown by the characteristic mobility shift after co-expression with WT IRAK1 (Fig. 4d), as demonstrated previously12, 13, 17. As expected, exogenously-expressed IRAK1 in Pin1 WT MEFs efficiently phosphorylated the IRAK1 N-terminal fragment, inducing the characteristic mobility shift (Fig 4d). However, like KD Irak1, WT Irak1 in Pin1 KO MEFs completely failed to induce any mobility shift of the N-terminal IRAK1 (Fig. 4d). These results together indicate that Pin1 is required for IRAK1 activation.


Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Tun-Kyi A, Finn G, Greenwood A, Nowak M, Lee TH, Asara JM, Tsokos GC, Fitzgerald K, Israel E, Li X, Exley M, Nicholson LK, Lu KP - Nat. Immunol. (2011)

Pin1 is essential for IRAK1 activation upon TLR ligation(a) Pin1 KO completely blocks IRAK1 activation in mouse cells following TLR7 stimulation. Pin1 WT and KO Flt3-derived pDCs (bottom) or TLR7-expressing MEF cells (top) were simulated with R-848 for the indicated times and analyzed for the characteristic IRAK1 shift by immunoblotting with IRAK1 antibodies, with IRAK4 and Pin1 amounts as controls.(b) Pin1 KO completely blocks activation of IRAK1, but not IRAK4 following TLR7 stimulation. Peritoneal macrophages from Pin1 WT and KO mice were stimulated with R-848 for the indicated times and kinase activity of IRAK1 and IRAK4 was assessed by an IP kinase autophosphorylation assay. IRAK1, IRAK4 and Pin1 protein were assayed as controls. (c) Pin1 knockdown blocks IRAK1 activation in human cells following TLR7 and TLR9, but not TLR3 stimulation. Human THP1 monocytes were infected with viral control shRNA or shRNA targeting Pin1 and simulated with poly (I:C) (TLR3), R-848 or CpG ligands for the indicated times, followed by analyzing the characteristic IRAK1 shift using immunoblotting.(d) In vivo kinase assay demonstrates IRAK1 kinase activity in Pin1 WT, but not Pin1 KO cells. Retroviral FLAG-IRAK1, and KD-IRAK1 or vector (VCT) control were coexpressed with a HA-N-terminal 220 aa fragment of IRAK1 as a substrate in Pin1 WT and KO MEFs (schematic diagram). IRAK1 kinase activity was determined by immunoblotting with HA antibodies to assess the characteristic mobility shift in IRAK1 N-terminal 220 aa due to trans-phosphorylation by co-expressed IRAK1 proteins.(e) Pin1 KO abolishes TLR dependent activation of exogenous IRAK1 in vivo. FLAG-IRAK1 and its KD mutant were co-expressed with TLR7 in Pin1 WT and KO MEF cells using retroviral vectors and stimulated with R-848 for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting.(f) Pin1, but not its WW domain-binding mutant (W34A) or catalytically inactive PPIase domain mutant (K63A), fully rescues IRAK1 activation in Pin1 KO cells. Pin1 KO MEFs stably expressing FLAG-IRAK1 were transfected with either WT-Pin1, K63A-Pin1, W34A-Pin1 or PPIase domain of Pin1 and TLR7 and stimulated for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. Results are representative of at least three independent experiments.
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Figure 4: Pin1 is essential for IRAK1 activation upon TLR ligation(a) Pin1 KO completely blocks IRAK1 activation in mouse cells following TLR7 stimulation. Pin1 WT and KO Flt3-derived pDCs (bottom) or TLR7-expressing MEF cells (top) were simulated with R-848 for the indicated times and analyzed for the characteristic IRAK1 shift by immunoblotting with IRAK1 antibodies, with IRAK4 and Pin1 amounts as controls.(b) Pin1 KO completely blocks activation of IRAK1, but not IRAK4 following TLR7 stimulation. Peritoneal macrophages from Pin1 WT and KO mice were stimulated with R-848 for the indicated times and kinase activity of IRAK1 and IRAK4 was assessed by an IP kinase autophosphorylation assay. IRAK1, IRAK4 and Pin1 protein were assayed as controls. (c) Pin1 knockdown blocks IRAK1 activation in human cells following TLR7 and TLR9, but not TLR3 stimulation. Human THP1 monocytes were infected with viral control shRNA or shRNA targeting Pin1 and simulated with poly (I:C) (TLR3), R-848 or CpG ligands for the indicated times, followed by analyzing the characteristic IRAK1 shift using immunoblotting.(d) In vivo kinase assay demonstrates IRAK1 kinase activity in Pin1 WT, but not Pin1 KO cells. Retroviral FLAG-IRAK1, and KD-IRAK1 or vector (VCT) control were coexpressed with a HA-N-terminal 220 aa fragment of IRAK1 as a substrate in Pin1 WT and KO MEFs (schematic diagram). IRAK1 kinase activity was determined by immunoblotting with HA antibodies to assess the characteristic mobility shift in IRAK1 N-terminal 220 aa due to trans-phosphorylation by co-expressed IRAK1 proteins.(e) Pin1 KO abolishes TLR dependent activation of exogenous IRAK1 in vivo. FLAG-IRAK1 and its KD mutant were co-expressed with TLR7 in Pin1 WT and KO MEF cells using retroviral vectors and stimulated with R-848 for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting.(f) Pin1, but not its WW domain-binding mutant (W34A) or catalytically inactive PPIase domain mutant (K63A), fully rescues IRAK1 activation in Pin1 KO cells. Pin1 KO MEFs stably expressing FLAG-IRAK1 were transfected with either WT-Pin1, K63A-Pin1, W34A-Pin1 or PPIase domain of Pin1 and TLR7 and stimulated for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. Results are representative of at least three independent experiments.
Mentions: Given that Pin1 binds to and isomerizes multiple pSer-Pro motifs in IRAK1 upon TLR activation, a key question is whether Pin1 regulates IRAK1 function in TLR signaling. Therefore, we examined the effects of Pin1 KO on IRAK1 activation in response to activation of various TLRs using Pin1 WT and KO MEFs and pDCs. Although TLR7 and TLR9 ligation activated IRAK1 in a time-dependent fashion in both Pin1 WT cells (Fig. 4a), as indicated by the mobility shift and increased kinase activity (Fig 4b), as previously described12, 13, 17, there was no evidence for IRAK1 activation in either assay in Pin1 KO MEFs or pDCs (Fig. 4a, b) or in Pin1-silenced THP1 cells using RNAi (Fig. 4c). Moreover, Pin1 KO also completely abolished IRAK1 activation in response to ligation of other TLRs including TLR2 and TLR4 (Supplementary Fig. 7a, b). These effects were highly specific because Pin1 KO did not affect activation of the IRAK1 upstream kinase IRAK4 (Fig. 4b), or mitogen-activated protein kinases (MAPK) including ERKs, JNKs and p38 MAPKs upon TLR activation (Supplementary Fig. 8). Similar observations were made following LPS stimulation of macrophages (Supplementary Fig. 9a). We also assessed the effects of Pin1 deficiency on IκB degradation following pDC stimulation with R-848 and CpG or treatment of macrophages with LPS and did not see any obvious difference between Pin1 WT and KO cells (Supplementary Fig. 9b, c). To further confirm this effect of Pin1 on IRAK1 activation, we developed an assay to measure the kinase activity of IRAK1 in cells utilizing the fact that IRAK1 can phosphorylate the N-terminal 220 amino acid IRAK1 fragment containing the UD in trans, as shown by the characteristic mobility shift after co-expression with WT IRAK1 (Fig. 4d), as demonstrated previously12, 13, 17. As expected, exogenously-expressed IRAK1 in Pin1 WT MEFs efficiently phosphorylated the IRAK1 N-terminal fragment, inducing the characteristic mobility shift (Fig 4d). However, like KD Irak1, WT Irak1 in Pin1 KO MEFs completely failed to induce any mobility shift of the N-terminal IRAK1 (Fig. 4d). These results together indicate that Pin1 is required for IRAK1 activation.

Bottom Line: Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms.The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown.We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Show MeSH
Related in: MedlinePlus