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 7

Figure 7. Model of the non-catalytic mechanism by which A20 limits IKK activation. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

Binding of IL-1β to IL-1R activates the E3 ligase TRAF6, which works with UBC13 to synthesize K63-linked polyubiquitin chains. The TAK1 and IKK complexes are recruited through ubiquitin binding domains on TAB2 and NEMO, respectively, allowing IKK phosphorylation by TAK1. A20 is recruited to NEMO through bipartite binding to polyubiquitin chains and an N-terminal region of NEMO. Formation of this complex blocks IKK phosphorylation by TAK1, thereby inhibiting IKK.

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
2.
Figure 5

Figure 5. Polyubiquitin chains directly and non-covalently induce specific binding between NEMO and A20. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) GST or GST-NEMO was incubated with affinity-purified A20 in the presence or absence of K63 polyubiquitin chains. After 15 min, 10% of input was withdrawn, and the remaining mixture was incubated with Glutathione Sepharose. Input and glutathione pull-down were immunoblotted using the indicated antibodies. (B) as in (A) except that, in lanes 6 & 7, polyUb was treated with isopeptidease T (IsoT). (C) As in (A), but including GST-NEMO ΔN. (D) Model of the polyubiquitin-NEMO-A20 complex. NT: N-terminus. NUB: NEMO ubiquitin binding. ZnF: zinc finger. Ub: ubiquitin.

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
3.
Figure 6

Figure 6. A20 inhibits IKK phosphorylation by TAK1 through a direct, non-catalytic mechanism. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) HEK293 cells were untreated or treated with TNFα for 10 min, and a NEMO antibody was used for immunoprecipitation. Immunoprecipitate was incubated with affinity-purified A20, then washed and incubated with γ–32P-ATP and GST-IκBα N-terminus or immunoblotted with the indicated antibodies. (B) as in (A), but NEMO immunoprecipitate was treated with vOTU or lambda phosphatase. Immunoprecipitate was immunoblotted for RIP1, p-IKK, or IKKβ. Aliquots of the immunoprecipitates were also assayed for IKK activity by measuring IκBα phosphorylation. (C) Method to generate constitutively active TAK1 (TAK1-ca). (D) TAK1 or TAK1-ca was incubated with IKK complex (top) or His6-MKK6 (K82A) (bottom), −/+ polyubiquitin chains and ATP. Samples were immunoblotted for p-IKK or p-MKK6. (E) Top and middle: as in (D), except that A20 was included in the reactions. Bottom: MEKK1 was used to activate IKK. (F) as in (D) top, except that the reactions included A20 WT, C103A, or C103A/ΔZnF7.

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
4.
Figure 1

Figure 1. A20 inhibits TNFα-induced IKK activation without antagonizing RIP1 ubiquitination. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) HEK293 cells stably overexpressing A20 and the parental (WT) HEK293 cells were stimulated with TNFα for the indicated time, then IKK complex was immunoprecipitated with a NEMO antibody for kinase assay using GST-IκBα N-terminus and γ-32P-ATP as substrates. Immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. N.S.: non-specific. (B) TNF receptor complexes were pulled down using glutathione Sepharose following stimulation of cells with GST-TNFα, and the proteins in the complexes were detected by immunoblotting with the indicated antibodies. (C) Similar to (B), except that a NEMO antibody was used for immunoprecipitation. (D) Parental HEK293 cells (WT) or HEK293 cells stably expressing A20 WT or A20 C103A with N-terminal TAP tag and C-terminal Flag tag were treated with TNFα for the indicated timepoints. Cell lysates were used for immunoblotting with the indicated antibodies.

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
5.
Figure 4

Figure 4. Polyubiquitin chains induce NEMO-A20 interaction. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) HeLa cells were stimulated with IL-1β for the indicated time, then immunoprecipitation was performed with a NEMO antibody. Immunoprecipitate was used to measure IKK activity as in Figure 1A, or for immunoblotting with an A20 antibody. (B) S100 was incubated with ATP, −/+ TRAF6, −/+ the IKK inhibitor TPCA-1 (10 μM), for 1 hr. A NEMO antibody was used for immunoprecipitation. Proteins in the S100 and immunoprecipitate were detected by immunoblotting with the indicated antibodies. (C) As in (B), following depletion of UBC13 and/or addition of vOTU or (D) A20 WT or F770A/G771A was added to S100. Experiment was His6-UBC13. performed as in (B) but without TPCA-1. (E) shUb-Ub (WT) or shUb-Ub (K63R) cells were untreated or treated with 1 μg/ml tetracycline for 4 days. Cells were then treated with TPCA-1 (20 μM) for 3 hr to enhance NEMO-A20 association. Cells were stimulated with IL-1β for the indicated times, then a NEMO antibody was used for immunoprecipitation. Proteins in the S100 and immunoprecipitate were detected by immunoblotting with the indicated antibodies. (F) MEF stable cell lines were treated for the indicated times with TNFα. Cell lysates were used for immunoprecipitation with a NEMO antibody. Cell lysate and immunoprecipitate were immunoblotted with the indicated antibodies.

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
6.
Figure 3

Figure 3. The roles of A20’s catalytic Cys 103 residue and ubiquitin binding in downregulation of NF-κB activity. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) Lysates from cells stably expressing A20 or GFP as a control were examined for A20 expression by immunoblotting. (B) Cells were untreated or treated with TNFα for 3 hr. RNA was extracted, and RT-PCR was performed to measure the relative expression of IL-6 or COX-2 as described in Experimental Procedures. Error bars indicate the standard error of the mean (SEM) among triplicates for each sample. Each graph is representative of three independent experiments. (C & D) Cells were treated for the indicated times with TNFα. Cell lysates were immunoblotted with the indicated antibodies; or immunoblotting was performed following immunoprecipitation with a NEMO antibody (D).

Brian Skaug, et al. Mol Cell. ;44(4):559-571.
7.
Figure 2

Figure 2. Cys 103 is dispensable, while ZnF7-dependent polyubiquitin binding is important for IKK inhibition by A20. From: Direct, Non-catalytic Mechanism of IKK Inhibition by A20.

(A) Left, silver stain of two-step affinity-purified A20. HSP70, 70kDa heat shock protein. Right, different amounts of purified A20 (WT and C103A) were incubated with HeLa S100, followed by addition of TRAF6 and ATP and further incubation for 1 hr. IKK activation in S100 was measured by immunoblotting for IκBα. (B) Domain architecture of A20, including relative locations of point mutations. OTU: ovarian tumor. ZnF: zinc finger. (C–E) Flag-A20 mutants were expressed in HEK293 cells and affinity purified. After incubation with HA-tagged polyubiquitin chains, A20 proteins were immunoprecipitated with a Flag antibody and ubiquitin chains were immunoblotted with an HA antibody. (F) IKK activation in S100 was measured as in (A). CC(AA): C779A/C782A. FG(AA): F770A/G771A. (G) MEF cells were infected with retrovirus expressing either GST or Flag-A20 WT, C103A, or F770A/G771A. The cells were stimulated with IL-1β, and then IKK activity was measured as in Figure 1A. Fold activation indicates activity normalized to the activity of unstimulated WT (A20+/+) cells (lane 1).

Brian Skaug, et al. Mol Cell. ;44(4):559-571.

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