Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases

Abstract

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88(E52A)) and 58 (MyD88(Y58A)) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88(K95A)) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27-72)), comprising the Glu(52) and Tyr(58) residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-kappaB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27-72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-kappaB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.

FIGURE 1.

Effect of mutations in MyD88 on NF-κB signaling in HeLa cells. A, alignment of MyD88 DD with homologous protein domains. Conserved amino acids of the hydrophobic core are shown in open boxes; similar polar or hydrophobic residues are displayed in gray, whereas conserved Ser/Thr residues are shown in light gray; underlined residues form α-helical motifs according to NMR or x-ray structures; residues in boldface type are the ones selected for mutagenesis studies. B, NF-κB activity monitored by luciferase reporter assay. HeLa cells were transiently transfected with wild type (WT) or mutant MyD88 constructs together with NF-κB reporter constructs expressing firefly luciferase and a control plasmid expressing Renilla luciferase. After 24 h, the cells were harvested, and luciferase activity was measured in soluble extracts as described under “Experimental Procedures.” Data were normalized for transfection efficiency by dividing firefly luciferase activity by that of the Renilla luciferase. Data are expressed as a percentage of wild type ± S.D. from a minimum of three separate experiments. Statistical significance was determined by Student's t test. *, p < 0.01; **, p < 0.05. C, expression levels of wild type or mutated MyD88 in transfected HeLa determined by Western blotting with either anti-FLAG- or anti-Myc-specific antibodies. Cell extract loading was normalized with anti-Erk2 antibodies (lower panel).

FIGURE 2.

Mutations in MyD88 DD affect protein self-association and recruitment of IRAK1 and IRAK4. A, HEK293T cells were transfected with Myc-MyD88 in combination with wild type (first lane) or mutated (second through sixth lanes) FLAG-MyD88. Twenty h after transfection, cells were collected, and self-association of MyD88 was assessed by co-immunoprecipitation. Cell extracts were immunoprecipitated (I.P.) with anti-FLAG antibodies, and the immunoprecipitated proteins were then analyzed by Western blotting with either anti-FLAG- or anti-Myc-specific antibodies to detect association. The mutations in FLAG-MyD88 increase MyD88 self-association about 2.5 times in comparison with the wild type protein. B, densitometric analysis of the effect of MyD88 mutants used in A on MyD88 dimerization. C–E, HEK293T cells were transfected with either Myc-IRAK1KD (C) or Myc-IRAK4KD (E) in combination with wild type (first lane in C and E) or mutated (second through sixth lanes in C and E) FLAG-MyD88. Twenty h after transfection, the cells were harvested, and the interaction of MyD88 with IRAKs was determined by co-immunoprecipitation. Cell extracts were immunoprecipitated with anti-FLAG antibodies, and the immunoprecipitated proteins were then analyzed by Western blotting with either anti-FLAG- or anti-Myc-specific antibodies to reveal the interaction. MyD88 mutants (E52A/E53A, E52A, and Y58A) strongly interfere with recruitment of IRAK1 and IRAK4 by MyD88 (densitometric analysis, D and F). Densitometric data in B, D, and F are expressed as a percentage of the wild type ± S.D. from a minimum of three separate experiments. Statistical significance was determined by Student's t test. *, p < 0,01; **, p < 0.05.

FIGURE 3.

Chimeric GFP-MyD88 proteins differentially affect NF-κB signaling. A, GFP-MyD88 fusion proteins are schematically depicted. B, NF-κB activity monitored by luciferase reporter assay. HeLa cells were transfected with GFP or GFP-MyD88 fusion protein constructs together with NF-κB reporter constructs expressing firefly luciferase and a control plasmid expressing Renilla luciferase. Twenty-four h after transfection, the cells were left untreated or were stimulated with either 30 ng/ml IL-1β (dark bars) or 100 μg/ml poly(I-C) (gray bars) for an additional 6 h. At the end of incubation, the cells were harvested, and luciferase activity was measured in soluble extracts as described under “Experimental Procedures.” NF-κB-driven firefly luciferase reporter activity was normalized to the control Renilla luciferase activity, as described above. Activities are plotted as percentage of GFP ± S.D. of at least three experiments for each fusion protein. GFP-MyD88-(27–72), GFP-MyD88-(30–66), and GFP-MyD88-(44–110) significantly reduced IL-1-dependent activation of NF-κB. *, p < 0.01 (n = 3). C, expression levels of GFP-MyD88 fusion proteins in transfected HeLa cells as determined by Western blot with anti-GFP antibodies. aa, amino acids.

FIGURE 4.

GFP-MyD88-(27–72) interferes with the subcellular localization of the p65/NF-κB protein. Immunofluorescence analysis of the effect of GFP-MyD88-(27–72) on nuclear translocation of endogenous NF-κB p65. HeLa cells were transfected with GFP or GFP-MyD88-(27–72) (panels E and F and panels G and H, respectively). Twenty h after transfection, the cells were left untreated (panels A, E, I, and O and panels C, G, M, and Q) or treated with 20 ng/ml IL-1β for 20 min (panels B, F, L, and P and panels D, H, N, and R). Cells were fixed, blocked, and stained with anti-NF-κB p65 antibodies (red) and Hoechst (blue) for nuclear staining. Nuclear NF-κB p65 is observed 20 min after treatment with IL-1β in the cells expressing GFP, but it is held back in the cytoplasm in those expressing GFP-MyD88-(27–72) (indicated by an arrow in D).

FIGURE 5.

GFP-MyD88-(27–72) interacts with IRAK1. A, HEK293T cells were transfected with GFP or GFP-MyD88-(27–72) in combination with Myc-IRAK4KD (lanes 1 and 2) or Myc-IRAK1KD (lanes 3 and 4) or Myc-MyD88 (lanes 5 and 6). Twenty h after transfection, the cells were collected, and the interaction of GFP-MyD88-(27–72) with Myc-IRAK4KD, Myc-IRAK1KD, or Myc-MyD88 was assessed by co-immunoprecipitation. Cell extracts were immunoprecipitated (IP) with anti-GFP antibodies, and immunoprecipitated proteins were analyzed by Western blot with either anti-GFP or anti-Myc antibodies to detect association. GFP-MyD88-(27–72) strongly interacts with IRAK1 (lane 4). Densitometric analysis of the degree of interaction of GFP-MyD88-(27–72) with Myc-IRAK4KD, Myc-IRAK1KD, or Myc-MyD88, respectively, is shown and is expressed as -fold induction with respect to MyD88. Statistical significance of the effects observed is indicated as follows. *, p < 0.01 (n = 3). B, HEK293T cells were transfected with Myc-IRAK1KD in combination with GFP (lane 1), GFP-MyD88 (lane 2), or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were collected, and the interaction of GFP-MyD88 and GFP-MyD88-(27–72) with Myc-IRAK1KD was assessed by co-immunoprecipitation. Cell extracts were immunoprecipitated (IP) with anti-GFP antibodies, and immunoprecipitated proteins were analyzed by Western blot with either anti-GFP or anti-Myc antibodies. Densitometric analysis of the interaction of GFP-MyD88 and GFP-MyD88-(27–72) with Myc-IRAK1KD is shown and is expressed as -fold induction with respect to GFP-MyD88.

FIGURE 6.

GFP-MyD88-(27–72) interferes with recruitment of IRAK1 and IRAK4 by MyD88. A, HEK293T cells were transfected with Myc-IRAK1KD alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of GFP (lanes 1 and 2) or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were collected, and the effect of either GFP or GFP-MyD88-(27–72) on the interaction of FLAG-MyD88 with Myc-IRAK1KD was evaluated by co-immunoprecipitation. Cell extracts were immunoprecipitated (IP) with anti-FLAG antibodies, and the immunoprecipitated proteins were then analyzed by Western blotting with either anti-FLAG or anti-Myc antibodies to detect association. GFP-MyD88-(27–72) strongly interferes with recruitment of IRAK1 by MyD88 (lane 3). Densitometric analysis of these results is depicted. GFP-MyD88-(27–72) significantly inhibited IRAK1KD recruitment by MyD88 (*, p < 0.01; n = 3). B, HEK293T cells were transfected with Myc-MyD88 alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of GFP (lanes 1 and 2) or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were harvested, and the effect of either GFP or GFP-MyD88-(27–72) on MyD88 self-association was assessed by co-immunoprecipitation. Cell extracts were immunoprecipitated with anti-FLAG antibodies, and immunoprecipitated proteins were analyzed by Western blotting with either anti-FLAG or the anti-Myc antibodies to detect MyD88 self-association. GFP-MyD88-(27–72) does not interfere with MyD88 self-association (lane 3). Densitometric analysis of these results is shown. C, HEK293T cells were transfected with Myc-IRAK1KD and Myc-IRAK4KD alone (lane 1) or in combination with FLAG-MyD88 (lanes 2 and 3) in the presence of GFP (lanes 1 and 2) or GFP-MyD88-(27–72) (lane 3). Twenty h after transfection, the cells were collected, and the effect of either GFP or GFP-MyD88-(27–72) on the interaction of FLAG-MyD88 with Myc-IRAK1KD and Myc-IRAK4KD was evaluated by co-immunoprecipitation. Cell extracts were immunoprecipitated with anti-FLAG antibodies, and the immunoprecipitated proteins were then analyzed by Western blotting with either anti-FLAG or anti-Myc antibodies to detect interaction. GFP-MyD88-(27–72) strongly interferes with recruitment of IRAKs by MyD88 (lane 3).

FIGURE 7.

GFP-MyD88-(27–72) interferes with activation of NF-κB induced by overexpression of IRAK1. A, NF-κB activity monitored by luciferase reporter assay. HeLa cells were transfected with GFP or GFP-MyD88-(27–72) together with NF-κB firefly luciferase, Renilla luciferase, and wild type (WT) Myc-IRAK1 (dark gray) or kinase-dead (KD) Myc-IRAK1 (light gray) constructs. Twenty-four h after transfection, the cells were harvested, and luciferase activity was measured in soluble extracts, as described under “Experimental Procedures.” NF-κB-driven firefly luciferase reporter activity was normalized to the control Renilla-luciferase activity, as described above. Activities are plotted as mean -fold induction ± S.D., compared with control only transfected with NF-κB firefly luciferase and Renilla luciferase constructs of at least three experiments. GFP-MyD88-(27–72) significantly reduces NF-κB activity induced by overexpression of Myc-IRAK1WT or Myc-IRAK1KD (*, p < 0.01; **, p < 0.05). B, expression levels of GFP, GFP-MyD88-(27–72) and Myc-IRAK1WT or Myc-IRAK1KD in transfected HeLa as determined by Western blotting with anti-GFP or anti-Myc antibodies. Cell extract loading was normalized with anti-tubulin (lower panel). GFP-MyD88-(27–72) exerts a dominant negative effect on IRAK1-induced activation of NF-κB.