Fig 1: Proposed model for IRAK-M activation mechanism by IRAK1 and PIN1, upon IL-33 stimulation. a IRAK-M is located in the Myddosome in association with IRAK1. PIN1 is phosphorylated at Ser71 and is inactive. b Upon IL-33 stimulation, IRAK1 is hyper phosphorylated and active and phosphorylates IRAK-M at S110P site. PIN1 is dephosphorylated and active. c PIN1 binds to IRAK-M phosphorylation site and induces conformational change that promotes its dissociation from the Myddosome. d Released IRAK-M translocates into the nucleus and induces the expression of its target genes for downstream TH2 cell differentiation and inflammation
Fig 2: IRAK-M expression is elevated in asthmatic human samples after Derp1 segmental allergic challenge. a Representative H&E staining and PAS staining of lung biopsies before and after Derp1 segmental challenge, as well as BALF and brush cytospins before and after treatment stained with Giemsa (n = 4). b Cytospin slides of brushing, total BALF cells, BALF CD15 + and CD205 + cells samples were immune stained for IRAK-M. c Quantification of IRAK-M expression in total BALF cells, BALF CD15 + and CD205 + cells samples as in b using Velocity program. d Total BALF cells were immune stained for PIN1 and pS71PIN1 and quantified using Velocity program e. f RNA was extracted from BALF cellular contents and analyzed for the relative expression of Il6, Csf3, Cxcl2 and Ccl5 by qRT-PCR. The data were analyzed by a Student’s two-tailed t test and the values are reported as mean ± standard errors of the means (SEM). *- statistical significance (P < 0.05), **- significance (P < 0.01). Scale bar = 50 µm
Fig 3: PIN1 binds to and isomerizes the pS110-P111 motif, located C-terminal to the IRAK-M DD. a Binding of the PIN1 WW domain to IRAK-M-pS110 peptide is demonstrated by overlaid regions extracted from 1H-15N HSQC spectra of 15N-labeled PIN1 WW domain that show progressive peak shifts with increasing peptide concentration (apo = red, purple = highest concentration). b Changes in chemical shift (??) with increasing IRAK-M-pS110 peptide concentration (filled circles) were fit to a simple bimolecular interaction model (solid lines) to yield the apparent dissociation constant, KDApp = 60.7 ± 11.5 µM (mean ± s.d.). c PIN1 catalysis of IRAK-M-pS110 and IRAK-M-S110E peptides is demonstrated by ROESY spectra of each peptide in the presence or absence of PIN1. In the presence of PIN1 ( + PIN1, top two spectra), cross peaks between cis and trans appear for both IRAK-M-pS110 and IRAK-M-S110E peptides. In the absence of PIN1 (-PIN1, bottom two spectra), no cross peaks were observed for either peptide. d 1H-15N HSQC spectrum of cleaved 15N-IRAK-M[1–119:R56D,Y61E]. e Ribbon diagram of the IRAK-M DD structure determined by NMR, showing the six alpha helices and highlighting the Y105-L20 interaction that anchors the C-terminal tail to the structure. f A ClusPro generated model of IRAK-M DD docked into a modified Myddosome oligomer containing three IRAK1 DD subunits in the L, M and N positions in the 3MOP structure. Blue: six MyD88 DD subunits, Green: Four IRAK4 DD subunits, Cyan: three IRAK1 DD homology model subunits, Yellow: docked NMR structure of the IRAK-M DD. g–j Proposed model for IRAK-M and PIN1 regulation of IRAK1 mediated immunity signaling. g IRAK1 homotetramer assembled on the Myddosome is able to achieve full hyperphosphorylation through each IRAK1 undefined domain (UD) being phosphorylated by neighboring IRAK1 kinase domains (KDs). h In the presence of IRAK-M, heterotetrameric IRAK-M/IRAK1 assembles on the Myddosome where IRAK-M DD replaces every-other IRAK1 DD subunit, preventing further IRAK1 hyper-phosphorylation and inhibiting IRAK1 release from the Myddosome thereby suppressing IRAK1-mediated inflammation. i IRAK1 KD phosphorylates S110 in next-neighbor IRAK-M. j Binding of PIN1 to IRAK-M pS110P (IRAK-M phosphorylated at residue S110) and subsequent isomerization induces release of IRAK-M from the Myddosome for downstream PIN1- and IRAK-M dependent signaling
Fig 4: IL-33 induces IRAK-M phosphorylation and binding to PIN1. a GST PIN1 pulldown assay with DC2.4 cell extracts either non-treated or treated with IL-33 (100 ng/ml) or LPS (100ng/ml) for 1 h. The GST-PIN1 bound proteins were eluted using reduced gluthatione and probed for IRAK-M. In the lower panel Coomassie blue staining of the blot shows equal amounts of GST or GST-PIN1 that were used for pull-down. b GST-PIN1 pulldown of DC2.4 cell extracts either treated or not with IL-33, followed by treatment in the absence or presence of calf intestinal alkaline phosphatase (CIP) for 30 min at room temperature before subjecting to GST-PIN1 pulldown. c DC2.4 cells were labeled with 10 µCi/ml {?-32 P}ATP for 3 h. The cells were washed with fresh medium and treated with 100 ng/ml IL-33 for the indicated times prior to IRAK-M immunoprecipitation. d DC2.4 cells stably expressing IRAK-M were treated with IL-33 and at the indicated time points were subjected to CO-IP using anti-PIN1 antibody and blotted for IRAK-M. e HEK293 cells were transfected with different IRAK-M constructs expressing the N’ terminal domain (aa1-220), the middle portion of the protein (aa220-440) or the C’ terminal domain (aa 440-630), and then treated with IL-33, followed by CO-IP for PIN1. f DC2.4 cells stably expressing IRAK-M were treated with IL-33 and subjected to GST or GST-PIN1 pull-down. The bound proteins were eluted and subjected to IP using IRAK-M antibody. g LC-MS/MS analysis shows phosphorylation of IRAK-M at position Ser110. h WT IRAK-M or its mutants lacking the death domain (IRAK-M ?DD), lacking the kinase domain (IRAK-M ?KD), IRAK-M S110A or IRAK-M S467A (where these serine residues were mutated to alanine) were expressed in HEK293 cells, followed by CO-IP for GFP after IL-33 treatment. i IRAK-M, S110E or P111A stably expressing DC2.4 cells were stimulated with IL-33 and PIN1 interaction was monitored by CO-IP experiment as indicated. j HEK293 cells were co-expressed with IRAK-M and GFP, GFP-PIN1, GFP-WW domain or GFP-PPIase domain and, then treated with IL-33 before CO-IP for GFP. k HEK293 cells were co-expressed with IRAK-M and WT PIN1, PIN1 mutant W34A, or PIN1 mutant K63A and then were treated with IL-33 before CO-IP for GFP
Fig 5: PIN1 increases IRAK-M protein stabilization. a After IRAK-M and GFP were coexpressed in WT and PIN1 KO MEFs for 24 h, cells were split equally into 5 dishes. 24 h later cells were treated with cyclohexamide and harvested to monitor IRAK-M, GFP and PIN1 levels at the time points indicated. b Quantification of 3 independent experiments as in a. c WT MEFs stably expressing the TET on inducible short hairpin for PIN1 (shPin1) or pLKO as a control, were expressed with IRAK-M, followed by induction with Doxycycline for 18 h prior to the cyclohexamide chase. d Quantification of 3 independent experiments as in c. e BMDCs from WT or PIN1 KO mice were treated with IL-33 and the levels of IRAK-M were monitored at different time points after induction. f Quantification of 3 independent experiments as in e. g PIN1 KO MEFs were expressed with IRAK-M alone or in combination with PIN1 or its mutants W34A or K63A for 24 h, followed by the cyclohexamide chase to assay IRAK-M stability. h Quantification of 3 independent experiments as in g. i IRAK-M or its different mutants; S110A, S110E and P111A were expressed in WT MEFs, followed by the cyclohexamide chase to assay IRAK-M stability. j Quantification of 3 independent experiments as in i. k IRAK-M or its different mutants were stably expressed in DC2.4 cells, followed by the cyclohexamide chase to monitor IRAK-M stability. l Quantification of 3 independent experiments as in k. The data were analyzed by a Student’s two-tailed t test and the values are reported as mean ± standard errors of the means (SEM). *- statistical significance (P < 0.05), **- significance (P < 0.01)
Supplier Page from MilliporeSigma for Anti-IRAK-M antibody produced in rabbit