Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 7

1.
Figure 3

Figure 3. Phosphorylated S131-, S144- and S173-Pro sites in the IRAK-1 UD bind to and are isomerized by Pin1. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a) Representative chemical shift perturbations in 15N-WW detected using 2D 15N-1H HSQC spectra resulting from titration with IRAK-1 peptides phosphorylated at Ser131, Ser144, and Ser173. Apo peaks are shown in red, and sequential colors represent increasing concentrations of peptides, purple being highest.
(b) Representative binding curves for WW domain residues, showing weighted chemical shift changes (Δδ = sqrt[Δδ1H2 + (0.154Δδ15N2]) as a function of total concentration of peptide. Residues plotted are S16 (●), S18 (×), Q33 (◆), the sidechain of W34 (■), and E35 (▲). Lines represent global fits.
(c) 2D 1H-1H ROESY spectra (mixing time of 100 ms) of IRAK-1 phosphopeptides in the presence (top panels) or absence (bottom panels) of a catalytic amount of Pin1. The appearance of exchange crosspeaks (arrows) between peaks corresponding to the cis and trans isomers confirms that Pin1 acts catalytically on these sequences.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
2.
Figure 5

Figure 5. Pin1 facilitates IRAK1 release from the receptor complex to activate IRF7 following TLR ligation. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a) Activated and phosphorylated IRAK1 is released from MyD88 in Pin1 WT cells, but inactive IRAK1 is not in Pin1 KO cells. HA-MyD88 and FLAG-IRAK1 were co-expressed in Pin1 WT and KO MEF using retroviral expression vectors, followed by immunoprecipitation with anti-HA antibody and then immunoblotting with anti-FLAG antibody.
(b) Pin1 knockdown inhibits the interaction of IRF7 with TRAF6. THP1 cells expressing Pin1-RNAi or control RNAi were stimulated with CpG for the indicated times and the interaction of IRF7 and TRAF6 was examined by Co-IP.
(c, d) Pin1 knockdown prevents IRF7 nuclear translocation in human THP1 cells. Following TLR7 (c) or TLR9 (d) ligation for the indicated times, nuclear and cytoplasmic fractions of THP1 cells were prepared, followed by immunoblotting with IRF7 antibody. The purity of nuclear and cytosolic fractions was evaluated by immunoblotting with tubulin or lamin A/C antibodies, respectively.
(e) Pin1 KO prevents IRF7 nuclear translocation after TLR7 or TLR9 ligation in pDCs. After R484 or CpG stimulation, Pin1 WT and KO pDCs were immunostained with IRF7 antibodies and counter-stained with DAPI, followed by confocal microscopy. Results are representative of at least three independent experiments.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
3.
Figure 7

Figure 7. Pin1 is required for TLR-mediated, type I interferon-dependent innate and adaptive immunity in vivo. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a-c) Pin1 KO mice completely fail to mount robust IFN-α response upon TLR7 or TLR9 activation. Pin1 WT and KO mice were injected with 50 nmol of R-848 (i.v.) (a), 5 μg CpG-A complexed to DOTAP (i.v.) (b), or MCMV 5× 104 PFU (i.p.) (c), followed by assaying serum IFN-α levels at different time points. (n=3)
(d, e) Pin1 KO mice are highly vulnerable to viral infection. Pin1 WT and KO mice were injected with 2.5×104 PFU MCMV, followed by monitoring changes in body weights over time (d) or with 105 PFU MCMV, followed by monitoring morbidity daily for 14 days (n=6) (e).
(f) Pin1 KO mice are severely defective in triggering the TLR-mediated, IFN-dependent adaptive immunity. Pin1 WT and KO mice were immunized with ovalbumin, anti-CD40 and CpG-A complexed to DOTAP and six days later, splenocytes were isolated and subjected to FACS analysis using antibodies against CD8a and CD44 and a MHC tetramer. The data shown were gated on CD8a-positive events and are representative of three independent experiments. The numbers indicate the percentage of tetramer-positive cells relative to the total number of CD8a+ T cells.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
4.
Figure 1

Figure 1. Pin1 is activated and required for cytokine and especially type I IFN secretion following TLR stimulation. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a-c) Impaired TLR7- and TLR9-induced cytokine production from Pin1 KO mDCs. Bone-marrow-derived mDCs were stimulated with 100 ng/ml LPS, 1 μg/ml Pam3CSK4, 0.1 μg/ml R-848 or 0.1 μM CpG-B. Concentrations of IL-6 (a), IL-12p40 (b), TNF (c) measured in cell-culture supernatants after 12 h are shown.
(d, e) IFN-α concentration in supernatants after R-848 and CpG-A treatment of purified splenic pDCs (B220+CD11cint) (d) and Flt3L-induced bone-marrow-derived pDCs for 24 h (e).
(f, g) IFN-α levels in supernatants following stimulation of splenic (f) and Flt3L-induced bone-marrow-derived pDCs (g) for 24 h with Influenza A (H1N1) virus or MCMV. IFN-α concentrations were measured by ELISA. Bars indicate means ±s.d. of triplicate determinations.
(h) Splenic pDCs were stimulated with PBS, R-848 or CpG DNA for 6 h. Expression of IFN-α or β mRNAs was measured by quantitative real-time RT–PCR analysis. Data were normalized to the levels of Gapdh expression. Results shown are means ± s.d. of triplicates.
(i) Pin1 catalytic activity, but not protein level, is increased upon TLR7 or TLR9 stimulation. Purified human PBMC were treated for 30 min either with PBS (▲), R-848 (■) or CpG DNA (△) and lysed, followed by protease-coupled isomerase activity assay for Pin1 activity. Results are representative of 3 independent experiments. Following the Pin1 protease coupled isomerase activity assay, fractions of lysates were subjected to immunoblotting analysis using Pin1 antibody with tubulin as a control (inset).

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
5.
Figure 6

Figure 6. Pin1 is required for IRF7 activation and IFN-α production upon TLR ligation in vitro. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS(GAL)-reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency.
(c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS(GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d), with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d).
(e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS(Gal), MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization.
(f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS(GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g). Expression levels of IRAK1 and its various mutants are shown below the graph (f).
(h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h), with representative pictures of VSV plaques shown in (i). ND, not detectable. Results shown are means ± s.d. of triplicates.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
6.
Figure 4

Figure 4. Pin1 is essential for IRAK1 activation upon TLR ligation. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(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.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.
7.
Figure 2

Figure 2. Proteomic approach identifies IRAK1 as a major Pin1 substrate upon TLR stimulation. From: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

(a) Proteomic identification of IRAK1 as a TLR-induced Pin1 binding protein. THP1 cells stimulated with R-848 for 45 min were lysed and subjected to GST-Pin1 pulldown followed by SDS-PAGE and colloidal CBB staining. Specific GST-Pin1 interacting bands were excised and 7 peptides were identified to IRAK1 by LC-MS (Supplementary Fig. 3a).
(b) TLR-dependent interaction between Pin1 and IRAK1, assayed by GST-Pin1 pulldown. RAW264.7 cells stimulated with PBS or either R-848 or CpG for 30 min were subjected to immunoblotting analysis using IRAK1 antibodies after pulldown with GST or GST-Pin1.
(c) TLR-dependent interaction between endogenous Pin1 and IRAK1, assayed by Co-IP. THP1 cells were stimulated with poly(I:C), R-848 or CpG and subjected to immunoprecipitation with anti-Pin1 antibodies or control IgG, followed by immunoblotting with IRAK1 antibodies.
(d) The IRAK1-Pin1 interaction is sensitive to phosphatase treatment. TLR7-HEK293T cells were transfected with FLAG-IRAK1 and stimulated with R-848 and lysates were untreated or treated with calf intestinal phosphatase (CIP) phosphatase for 60 min at 30°C, followed by GST-Pin1 pulldown experiments.
(e) The Pin1-IRAK1 interaction is dependent on the intrinsic kinase activity of IRAK1. FLAG-KD-IRAK1, either alone or in combination with IRAK1 were expressed in IRAK1-deficient 293T cells, followed by GST pulldown experiments
(f) Pin1 binds directly to phosphorylated WT IRAK1, but not KD IRAK1. FLAG-IRAK1 and FLAG-KD IRAK1 were expressed in IRAK1-deficient 293T cells and purified using FLAG-agarose, followed by Far-Western analysis using GST-Pin1 WW domain to detect Pin1 binding using anti-GST antibody. Membranes were re-probed with FLAG antibody as a control.
(g) Pin1 binds to activated WT IRAK1, but not KD IRAK1 in MEFs. FLAG-IRAK1 and its KD mutant were expressed in MEFs using retroviral infection and then treated with R-848 or control buffer, followed by GST pulldown experiments.
(h) Multiple pSer-Pro motifs in the undetermined domain (UD) of IRAK1 are required for Pin1 binding. FLAG-IRAK1 and its mutants were expressed in MEFs using retroviral infection, and then treated with R-848 or control buffer, followed by GST pulldown experiments.
(i) S173 phosphorylation of IRAK1 is induced upon TLR7 and TLR9 stimulation. THP1 cells were stimuylated with CpG or R-848 and thereafter intracellularly stained with an anti-pS173 antibody, followed by a secondary FITC conjugated antibody. Fluorescence was measured by flow cytometry.

Adrian Tun-Kyi, et al. Nat Immunol. ;12(8):733-741.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk