SCFbeta-TrCP ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase

Abstract

Processing of the p105 precursor to form the active subunit p50 of the NF-kB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkB kinase (IkK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918±934) serves as a recognition motif for the SCF b-TrCP ubiquitin ligase. Expression of IkKb dramatically increases processing of wild-type p105, but not of p105-D918±934. Dominant-negative b-TrCP inhibits IkK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-D918±934 associate physically following phosphorylation. In vitro, SCF b-TrCP speci®cally conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkBs, b-catenin and human immunode®-ciency virus type 1 Vpu. Since p105-D918±934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. Keywords: IkB kinase (IkK)/NF-kB/p105/b-TrCP/ ubiquitin IntroductionThe NF-kB transcription factors play key roles in basic processes such as regulation of the immune and in¯am-matory responses, development and differentiation, malignant transformation and apoptosis (Baeuerle and Baltimore, 1996;Baldwin, 1996;Barnes and Karin, 1997;Ghosh et al., 1998;Foo and Nolan, 1999). The precursor molecules p105 and p100 undergo ubiquitinand proteasome-mediated limited processing to yield the respective active subunits p50 and p52 (Palombella et al., 1994;Orian et al., 1995;Betts and Nabel, 1996), which are derived from the N-terminal domain of the molecule. The C-terminal domain is degraded (Fan and Maniatis, 1991). These subunits typically heterodimerize with members of the rel family, such as p65, RelB or c-Rel, to generate the active transcription factor. In the resting cell, the heterodimer generates a ternary complex with a member of the IkB family of inhibitory proteins and is sequestered in the cytosol. Following stimulation, speci®c IkB kinases are activated (Mercurio et al., 1997;Woronicz et al., 1997;Zandi et al., 1997) and phosphorylate the protein on serine residues 32 and 36 (Brown et al., 1995). The phosphorylation leads to recognition of the molecule by the SCF b-TrCP ubiquitin ligase complex (see, for example, Yaron et al., 1998;Winston et al., 1999), polyubiquitylation on Lys21 and/or Lys22 and subsequent degradation by the 26S proteasome (Alkalay et al., 1995;Chen et al., 1995). Following degradation of IkBa, the heterodimer is translocated into the nucleus where it initiates speci®c transcription.The ubiquitin pathway is involved in the regulation of many basic cellular processes, such as cell cycle progression, differentiation and development, and the immune and in¯...

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Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

“…npg S932 in the p105 PEST region (see Figures 3 and 4). These phosphorylations induce the binding of SCF βTrCP E3 ligase, which catalyzes the subsequent K48-linked polyubiqitination and proteolysis of p105 by the proteasome [42,44,45]. This predominantly leads to the complete degradation of p105, rather than processing to p50 [42,44].…”

Cot/tpl2 activity is required for TLR‐induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression

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