the ability to activate NFkB in 293-I1A cells upon IL-1 stimulation. On the other hand, while E71A and Q78G exhibited partial defect, the combination of these two mutations greatly decreased the IL-1-induced NFkB-dependent luciferase activity in 293-I1A cells. The same results were obtained from the analysis of the IRAK-M mutants in NFkB electrophoretic mobility shift assay. Consistent with its ability in mediating IL-1-induced NFkB activation, IRAK-M was also able to 1702259-66-2 manufacturer restore IL-1-induced IL-8 and TNFa gene expression in 293I1A cells. Importantly, W74A and E71A/Q78G IRAK-M mutants failed to restore the IL-8 and TNFa gene expression in 293-I1A cells in response to IL-1 stimulation. Moreover, since we predict the hydrophilic part of Q78 in IRAK-M may interact with the backbone oxygen atoms of F25 in IRAK-4, we generated the IRAK-4 F25D mutant. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828810 When tested in IRAK-4-deficient cells, this F25D mutant showed decreased interaction with IRAK-M compared with wild-type IRAK-4 upon IL-1 stimulation, confirming the critical contact between Q78 in IRAK-M and F25 in IRAK-4. We then confirmed the binding interface of IRAK-M with IRAK-4 in response to TLR7 stimulation. IRAK-M wild-type and mutants were expressed in IRAK-1/2/M-TKO-BMDMs by adenovirus infection. Consistent with the results for the IL-1 response, wild-type IRAK-M was able to interact with IRAK-4 and restored TLR7-induced NFkB activation in IRAK-1/2/ M-TKO-BMDMs, whereas W74A mutant lost its ability to interact with IRAK-4 and failed to mediate NFkB activation. While E71 and Q78G showed partial reduction, the combinational mutant E71A/Q78G was unable to interact with IRAK-4 with abolished TLR7-induced NFkB activation. Furthermore, W74A and E71A/Q78G mutants also failed to restore CXCL1, TNFa and IL-6 gene expression in IRAK-1/2/ M-TKO-BMDMs upon TLR7 stimulation. Taken together, these results provide strong supporting evidence for the interaction between DDs of IRAK-M and IRAK-4 and demonstrate that this interaction is critical for IRAK-M to mediate IL-1R/TLR-induced NFkB activation. IRAK-M deficiency resulted in decreased second wave TLR7-induced NFjB activation in the presence of IRAK-1 and IRAK-2 
Previous studies suggested that IRAK-M functions as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibits all downstream signalling events including NFkB activation. Considering how IRAK-M can form Myddosome and is capable of mediating TLR/IL-1R signalling in the absence of IRAK-1/IRAK-2, we decided to re-investigate the impact & IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al of IRAK-M single deficiency on TLR7-mediated signalling. To our surprise, IRAK-M deficiency decreased TLR7-induced late phase NFkB activation in a gel-shift assay. The TAK1-dependent downstream signalling events were similar or slightly enhanced in IRAK- MKO-BMDMs compared to that in control cells. However, whereas TLR7-induced IkBa and p65 phosphorylation was comparable at early times, they was substantially reduced at later times . These results suggest that IRAK-M Myddosome contributes to the second wave of TLR7-induced NFkB activation in the pre- & 2013 European Molecular Biology Organization IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al sence of IRAK-1 and IRAK-2, probably through the TAK1independent pathway. Our previous studies showed that MG132, which inhibits proteasome-dependent protein degradati