Model of IL-1 signaling displaying the negative regulatory role of Pellino 3b. Upon IL-1 stimulation, adapter molecules MyD88 and Tollip are first recruited to IL-1 receptor, which in turn recruits IRAK4, IRAK, and TRAF6, resulting in the formation of the receptor complex (complex I). During the formation of complex I, IRAK4 is activated, leading to the hyperphosphorylation of IRAK, which creates an interface for the interaction of Pellino 1/2 with the IRAK4-IRAK-TRAF6 complex (Intermediate complex). Pellino 2 might mediate Lys-63-linked IRAK ubiquitination, leading the interaction of intermediate complex Pellino 2-IRAK4-IRAK-TRAF6 with the membrane-bound pre-associated TAK1-TAB1-TAB2, resulting in the formation of complex II (TAK1 complex, IRAK-TRAF6-TAK1-TAB1–TAB2). An unknown E3 then mediates Lys-48-linked IRAK ubiquitination, leading to degradation of IRAK, resulting in the translocation of TRAF6-TAK1-TAB1–TAB2 (complex III) from the membrane to the cytosol where TAK1 is activated, eventually resulting in the activation of NFκB. Pellino 3b is up-regulated upon IL-1 stimulation and interacts with the TAK1 complex (complex VI), where Pellino 3b mediates efficient Lys-63-linked IRAK ubiquitination, competing with Lys-48-linked IRAK ubiquitination, thereby inhibiting IRAK degradation and subsequent release of the complex III from the membrane to the cytosol, attenuating TAK1-dependent NFκB activation.

Pellinos can mediate either Lys-63- or Lys-48-type polyubiquitination in vitro. A, Pellino 3b and Pellino 2 act together with Ubc13/Uev1A to assemble polyubiquitin chain in vitro. Purified recombinant Pellino 1, Pellino 2, or Pellino 3b (1 μg) was incubated with 100 nm E1, 250 nm UbcH13/Uev1A or UbcH5, and 5 μg of ubiquitin at 30 °C for 1 h as described under “Materials and Methods.” Following in vitro ubiquitination assay, 20 μl of Laemmli buffer was added into 10 μl of reaction. 15 μl of the reaction mixture was analyzed by Western blotting (WB) with anti-ubiquitin, and the other half was visualized by Coomassie Blue staining after resolving by 10% SDS-PAGE. B, Pellino 3b and Pellino 2 assemble Lys-63-linked polyubiquitin chain. Recombinant Pellino 1, Pellino 2, or Pellino 3b was incubated with E1, UbcH13/Uev1A, and various mutant ubiquitin at 30 °C for 1 h. C, phosphorylated Pellino 1 acts with UbcH13/Uev1A to assemble the Lys-63-linked poly(Ub) chain. Recombinant Pellino 1 was incubated with 0.5 μg of purified IRAK, 100 nm E1, 500 nm UbcH13/Uev1A, and various mutant ubiquitin at 30 °C for 1 h. D, phosphorylated Pellinos act together with UbcH5 or UbcH3 to assemble polyubiquitin chains in vitro. Purified recombinant Pellino 1, Pellino 2, or Pellino 3b (1 μg) was incubated with 0.5 μg of purified IRAK, 100 nm E1, 1 μm UbcH5 or UbcH3, and 5 μg of ubiquitin at 30 °C for 1 h. E, phosphorylated Pellinos act with UbcH3 to assemble Lys-48-linked polyubiquitin chain. Purified recombinant Pellino 1, Pellino 2, or Pellino 3b (1 μg) was incubated with 0.5 μg of purified IRAK, 100 nm E1, 1 μm UbcH3, and 5 μg of wild type (WT) or mutant ubiquitin at 30 °C for 1 h. Following in vitro ubiquitination assay, 20 μl of Laemmli buffer was added into 10 μl of reaction. 15 μl of the reaction mixture was analyzed by Western blotting with anti-ubiquitin, whereas the other half was visualized by Coomassie Blue staining after resolving by 10% SDS-PAGE.

The RING finger-like motif is required for the E3 ligase activity of Pellino 3b. A, Pellino 3b and Pellino 2 promote self-ubiquitination in vivo. Cell lysates prepared for 293-IL-1R cells transfected with FLAG-tagged Pellino 1, Pellino 2, or Pellino 3b were immunoprecipitated (IP) with anti-FLAG followed by Western blotting (WB) with anti-ubiquitin and anti-FLAG, respectively. B, Pellino 2 and 3b differentially regulate NFκB activity. Various amounts (0–100 ng) of plasmids encoding Pellino 2 or Pellino 3b were co-transfected with Elam-luciferase reporter plasmid (Renilla luciferase reporter as control) into C6 cells. Following transfection for 20 h, cells were either left untreated or stimulated by IL-1 (1 ng/ml) for 6 h and then harvested for measuring luciferase activity. The relative luciferase units were counted by normalizing firefly luciferase activity against Renilla luciferase activity. The fold of induction was calculated for each sample as IL-1-induced relative luciferase units/nontreated relative luciferase units. C, RING-finger like domain is essential for the self-ubiquitination of Pellino 3b. Cell lysates prepared from 293-IL-1R cells transfected with wild-type (WT) Pellino 3b or Pellino 3b mutants were immunoprecipitated with anti-FLAG followed by Western analysis with anti-FLAG and ubiquitin. D, RING finger is essential for the E3 ligase activity of Pellino 3b. Purified Pellino 3b mutant (MT) protein (C422S/C425S) was incubated with E1, Ubc13/Uev1A, and ubiquitin to carry out in vitro ubiquitination assay as described above. E, IRAK was co-transfected with FLAG-tagged wild-type or mutants of Pellino 3b into I1A cells. Cell lysates from the transfected cells were denatured and immunoprecipitated with anti-IRAK-agarose, followed by Western analysis with anti-ubiquitin and anti-IRAK. Cell lysates were also directly analyzed by Western blotting with anti-FLAG.

Pellino 3b associates with TAK1-IRAK-TRAF6 (complex II) in IL-1 signaling. A, IL-1 induces the expression and modification of Pellino 3 in 293-IL-1R cells. Cells lysated from 293-IL-1R cells untreated or stimulated with 1 ng/ml IL-1 for the indicated times were analyzed by Western blotting with anti-Pellino 3 and anti-actin. B, association of endogenous Pellino 3 and IRAK. Cell lysates from 293-IL-1R cells stimulated with IL-1 (1 ng/ml) were immunoprecipitated (IP) with anti-Pellino 3, and followed by immune blotting with anti-IRAK, anti-TRAF6, and anti-TAK1, respectively.

IL-1-induced IRAK polyubiquitination is linked through both Lys-48 and Lys-63. 293-IL-1R cells transfected with HA-tagged wild-type (WT) ubiquitin or ubiquitin mutants (including Lys-63 only, K48R, Lys-48 only, and K63R as described in the text) were stimulated with 1 ng/ml of IL-1 for 15 min or left untreated, followed by immunoprecipitation (IP) with anti-IRAK and Western analyses with anti-HA and anti-IRAK, respectively.

Pellinos promote Lys-63-linked polyubiquitination of IRAK in vivo. A, IRAK was co-transfected with FLAG-tagged Pellino 1, Pellino 2, or Pellino 3b into I1A cells (IRAK-deficient). Cell lysates from the transfected cells were analyzed by Western blotting (WB) with anti-IRAK and anti-FLAG (A) or immunoprecipitated (IP) by anti-IRAK-agarose and followed by anti-ubiquitin and anti-IRAK blotting (B). C, IRAK was co-transfected with HA-tagged wild-type or mutants of ubiquitin into I1A cells, with FLAG-pellino 1 or 2. Cell lysates were denatured and immunoprecipitated with anti-IRAK-agarose, followed by Western blotting with anti-HA and anti-IRAK. Cell lysates were directly analyzed by Western blotting with anti-FLAG or anti-HA. D, IRAK was co-transfected with HA-tagged wild-type or mutants of ubiquitin into I1A cells, with or without FLAG-pellino 3b. Cell lysates were denatured and immunoprecipitated with anti-IRAK-agarose, followed by Western blotting with anti-HA and anti-IRAK. Cell lysates were directly analyzed by Western blotting with anti-FLAG or anti-HA.

Pellino 3b-mediated Lys-63-linked polyubiquitination inhibits IRAK degradation induced by IL-1. A, Pellino 3b ubiquitinates IRAK on Lys-134. FLAG-tagged wild-type (WT) or mutants (mt) of Pellino 3b were co-transfected with wild-type or K134R mutant IRAK into I1A cells. Cell lysates were analyzed directly by Western blotting (WB) with anti-IRAK and anti-FLAG or immunoprecipitated (IP) by anti-IRAK and followed by anti-ubiquitin and anti-IRAK blotting. B, Lys-134 mutant IRAK plasmid was co-transfected with wild-type or mutants of HA-ubiquitin, with or without FLAG-pellino 3b, into I1A cells. Cell lysates were denatured and immunoprecipitated by anti-IRAK. Immunoprecipitates were probed with anti-HA and anti-IRAK. Cell lysates were also blotted with anti-HA and anti-FLAG. C, knockdown of Pellino 3 enhances IRAK degradation in IL-1 signaling. Control clone (-, stably transfected with pSUPER-scrambled into 293-IL-1R cells) and two Pellino 3 knockdown clones (+, stably transfected with pSUPER-pellino 3) were stimulated with 1 ng/ml IL-1 for indicated times. The cells lysates were analyzed by Western blotting with anti-IRAK, anti-Pellino 3, and anti-actin, respectively. D, wild-type or mutants of HA-ubiquitin plasmids were transfected into control or Pellino 3 knockdown cells. Two days after transfection, cells were either treated with IL-1 for 15 min or left untreated. Cell lysates were denatured and immunoprecipitated by anti-IRAK and immunoblotted with anti-HA and anti-IRAK. Cell lysates were also directly blotted with anti-HA and anti-Pellino 3. E, Pellino 3b-mediated Lys-63-linked polyubiquitination competitively inhibits Lys-48-linked polyubiquitination of IRAK in IL-1 signaling. 293-IL-1R cells and 293-IL-1R cells stably transfected with FLAG-tagged Pellino 3b were transfected with HA-tagged wild-type or mutants of ubiquitin. The transfected cells were stimulated with 1 ng/ml IL-1 for 15 min or left untreated. Cell lysates were denatured and immunoprecipitated with anti-IRAK-agarose, followed by Western analysis with anti-HA and anti-IRAK. Cell lysates were analyzed by Western blotting with anti-FLAG and anti-HA. F, IL-1-induced IRAK degradation is abolished in FLAG-pellino 3b expressing clone. 293-IL-1R cells and 293-IL-1R cells stably transfected with FLAG-tagged pellino 3b were stimulated with 1 ng/ml of IL-1 for the indicated time. Cell lysates were analyzed by Western blotting with anti-IRAK and anti-actin. G and H, Pellino 2 and Pellino 3b exert different effects on IL-1-induced IRAK degradation. 293-IL-1R cells and 293-IL-1R cells stably transfected with FLAG-tagged Pellino 3b or Pellino 2 were stimulated with 1 ng/ml of IL-1 for the indicated time. Cell lysates were analyzed by Western blotting with anti-IRAK, anti-FLAG, and anti-actin, respectively. Anti-IRAK and anti-actin blots were quantified by densitometry, and the relative intensity (the weakest band of each blot was normalized as 1) is shown below the blot. Then the IRAK/actin ratio of each sample was calculated, and this is considered as the arbitrary IRAK protein amount presented in each sample. The arbitrary IRAK of the untreated samples of each cell line was set as 100%, and the relative arbitrary IRAK of the other samples (versus the arbitrary IRAK of the untreated sample from the same cell line) was calculated and plotted in H.

Pellino 3b negatively regulates TAK1-dependent signaling induced by IL-1. A, knockdown of Pellino 3 enhances IL-1-activated TAK1-IKK pathway. Pellino 3 knockdown and control cells were stimulated by 1 ng/ml IL-1 for the indicated time. Cell lysates were analyzed by Western blotting with anti-TAK1, anti-TAB1, anti-p-IKKα/β, anti-IKKα/β, anti-p-IκBα, anti-IκBα, anti-p-JNK, anti-JNK, anti-Pellino 3, and anti-actin. B, Pellino 3b inhibits IL-1-induced TAK1-IKK signaling. Control and FLAG-pellino 3b expressing cells were stimulated with 1 ng/ml IL-1 for the indicated time. Cell lysates were analyzed by Western blotting with anti-TAK1, anti-TAB1, anti-p-IKKα/β, anti-IKKα/β, anti-p-IκBα, anti-IκBα, anti-p-JNK, anti-JNK, anti-FLAG, and anti-actin. C, NFκB gel shift assay. Pellino 3 knockdown cells and Pellino 3b stably transfected cells, along with their control cells, were stimulated by IL-1 (1 ng/ml) for the indicated time periods. Cell lysates were incubated with ³²P-labeled NFκB gel shift oligonucleotides (sc-2511, Santa Cruz Biotechnology) and then resolved by 5% native polyacrylamide gel. D, E3 ligase activity is necessary for Pellino 3b to negatively regulate IL-1 signaling. The RING finger mutant (C422S/C425S) and wild-type (WT) Pellino 3b stably transfected cells were stimulated with 1 ng/ml IL-1 for the indicated time. Cell lysates were analyzed by Western blotting with anti-IRAK, anti-p-IKKα/β, anti-p-IκBα, anti-IκBα, anti-p-JNK, anti-FLAG, and anti-actin. E, Pellino 3b does not inhibit TNFα signaling. Control and FLAG-pellino 3b expressing cells were stimulated with 10 ng/ml TNFα for the indicated time. Cell lysates were analyzed by Western blotting with anti-p-IκBα, anti-IκBα, anti-p-JNK, and anti-actin. F, Pellino 3 negatively regulates IL-1-induced IL-8 production. Control and Pellino 3 knockdown cells were stimulated by 1 ng/ml IL-1 for 8 h or left untreated. Media were collected, and secreted IL-8 was measured by enzyme-linked immunosorbent assay. G, MG-132 inhibits IL-1-induced TAK1-IKK signaling. 293-IL-1R cells were untreated or treated with IL-1 with or without MG-132. Cell lysates were analyzed by Western blots with anti-IRAK, anti-TAK1, anti-p-IKKα/β, anti-IKKβ, anti-p-IκBα, anti-IκBα, and anti-actin.