Characterization of the IκB-kinase NEMO Binding Domain

Abstract

Proinflammatory activation of NF-B requires an upstream kinase complex (IB-kinase; IKK) composed of two catalytic subunits (IKK␣ and IKK␤) and a noncatalytic regulatory component named NEMO (NF-B essential modulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKK␤ interactions and inhibits tumor necrosis factor-␣-induced NF-B. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKK␣ and IKK␤, and mutational analysis of the IKK␤ NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKK␤ through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKK␤ NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex.

Inhibition of NF-κB Activation by Peptides Targeting NF-κB Essential Modulator (NEMO) Oligomerization

“…We found that the NF-B pathway was strongly inhibited when pre-B lymphocytes were preincubated with BA-CC2 (IC 50 ϭ 22 M) or BA-LZ (IC 50 ϭ 3 M). The latter value is about 2 orders of magnitude lower than that of the previously described pep- tide used to disrupt the NEMO/IKK interface (13,37). Similar values were obtained when we assayed the DNA binding capacity of NF-B using the TransAM TM NF-B p65 Chemi kit provided by Active Motif Inc. (data not shown).…”

“…In a previous study, a cell-permeable peptide corresponding to the C terminus of IKK-␣ and IKK-␤ was used to compete with the binding of NEMO, thereby preventing the assembly of the IKK complex and blocking activation of the kinases (13,37). Other cell-permeable peptides have been designed to interfere with the NF-B pathway.…”

Amelioration of acute inflammation by systemic administration of a cell‐permeable peptide inhibitor of NF‐κB activation

“…The housekeeping gene ␤-actin was used as an internal control. Total RNA (7 g) was reverse-transcribed into complementary DNA by using oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). Complementary DNA (1 l) was amplified by PCR using Taq polymerase (Promega, Madison, WI) according to the manufacturer's instructions.…”

“…Investigators in our group have previously reported that an NH 2 -terminal ␣-helical region of NEMO associates with a hexapeptide sequence within the extreme COOH-terminus of both IKK␣ and IKK␤, and we have named this region the NEMO-binding domain (NBD) (12,13). Our group also found that a short cell-permeable peptide spanning the IKK␤ NBD, which we named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNF␣-induced NF-B activation in vivo.…”

Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity

“…Plasmids and Antibodies-Expression vectors encoding FLAG-tagged human full-length NEMO, NEMO , NEMO 1-100 , wild type and dominant negative IKK␤, Xpress-IKK␣, and Xpress-IKK␤ have been described elsewhere (26). The 3xB firefly luciferase reporter plasmid and the Renilla luciferase reporter plasmid were described previously (27).…”