betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932 - PubMed (original) (raw)

betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932

Valerie Lang et al. Mol Cell Biol. 2003 Jan.

Abstract

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.

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Figures

FIG. 1.

FIG. 1.

IKK target sequences, p105 point mutants, and synthetic phosphopeptides. (A) Schematic representation of mammalian NF-κB1 p105. The relative positions of the Rel homology domain (RHD), nuclear localizing signal (NLS), glycine-rich region (GRR), ankyrin repeats (vertical rectangles), death domain (DD), and PEST domain are shown. An alignment of the IKK target sequence in the p105 PEST region with those in IκBα, IκBβ, and IκBɛ is also shown (all human proteins). (B) Serine-to-alanine point mutants of HA-p105. WT, wild type. (C) Sequences of synthetic p105 and IκBα peptides. P, phosphorylated residue.

FIG. 2.

FIG. 2.

Serine 932 is essential for TNF-α to trigger proteolysis of p105. (A) Clones of HeLa cells stably transfected with HA-p105, HA-p105(S923A), HA-p105(S927A), or HA-p105(S932A) were metabolically pulse-labeled with [35S]methionine-[35S]cysteine (45 min) and then chased for the indicated times in complete medium (control) or complete medium supplemented with TNF-α. Anti-HA immunoprecipitates were resolved by SDS-7.5% PAGE and revealed by fluorography. Amounts of immunoprecipitated wild-type (WT) and point-mutated HA-p105 were quantified by laser densitometry (mean ± standard error of the mean; n = 5). (B) HeLa cells stably transfected with either HA-p105 or HA-p105(S932A) were analyzed by pulse-chase metabolic labeling as described for panel A. Anti-HA immunoprecipitates (Ip) were resolved by SDS-7.5% PAGE and revealed by fluorography. The position of HA-p105 is indicated with an arrow. (C) Cell lysates from the indicated HeLa clones with or without TNF-α stimulation (15 min) were Western blotted for endogenous IκBα. (D) Stably transfected HeLa cells were preincubated for 1 h with LLnL proteasome inhibitor and then stimulated for 15 min with TNF-α or control medium. Immunoprecipitates of wild-type and point-mutated HA-p105 were then sequentially Western blotted with the indicated antibodies.

FIG. 3.

FIG. 3.

TNF-α induces rapid phosphorylation of p105 on serine 932. (A) NIH 3T3 cells were transiently cotransfected with expression vectors encoding wild-type (WT) HA-p105 or the indicated mutants and IKK2 or EV and treated with MG132 proteasome inhibitor for the last 4 h of a 48-h culture. HA-p105 was immunoprecipitated, and isolated protein was sequentially Western blotted with anti-phospho-Ser932 (upper gel), anti-phospho-Ser927 (middle gel), and anti-p105C (lower gel) antibodies. Ip, immunoprecipitates. (B) HeLa cells were preincubated for 30 min with MG132 and then stimulated for the indicated times with TNF-α or control medium (C). Immunoprecipitates of endogenous p105 were then Western blotted sequentially with the indicated antibodies. (C) HeLa cells stably transfected with the indicated HA-p105 constructs were preincubated for 1 h with LLnL inhibitor and then stimulated for 15 min with TNF-α or control medium. Anti-HA immunoprecipitates were then sequentially Western blotted with the indicated antibodies.

FIG. 4.

FIG. 4.

The IKK complex directly phosphorylates serine 932 of p105. (A) Wild-type (WT) and IKK1/2-/- (−/−) EFs were pretreated with MG132 for 30 min and then incubated for a further 30 min with TNF-α or control medium. p105 was then immunoprecipitated from cell lysates and Western blotted with the indicated antibodies. Ip, immunoprecipitates. (B) IKK1/2−/− or wild-type EFs were transiently cotransfected with the indicated expression vectors. Cells were treated with MG132 for the last 4 h of a 48-h culture. HA-p105 was isolated from cell lysates by immunoprecipitation and immunoblotted with the antibodies shown. (C) In vitro kinase assays were carried out with baculovirus-produced purified His6-IKK1 or His6-IKK2 (100 ng), using as substrates 5 μg of wild-type or point-mutated GST-p105758-967 protein, as indicated. Phosphorylation was assessed by sequentially immunoblotting with anti-phospho-Ser932 (upper gel) or anti-phospho-Ser927 (middle gel) antibodies. Equal loading of fusion proteins was confirmed by reprobing blots with anti-GST MAb (lower gel). (D) HeLa cells were stimulated for 15 min with TNF-α or left unstimulated. The endogenous IKK complex was isolated from cell lysates by immunoprecipitation with an anti-NEMO antibody. Control immunoprecipitations were carried out with nonimmune rabbit serum (NRS). Phosphorylation was assessed as described for panel C. Blots were probed with anti-NEMO antibody to confirm that equal amounts of IKK complex were immunoprecipitated.

FIG. 5.

FIG. 5.

Serines 927 and 932 are required for TNF-α to trigger p105 ubiquitination. (A) HeLa cells stably transfected with HA-p105 were pretreated for 1 h with LLnL proteasome inhibitor or vehicle control (dimethyl sulfoxide [DMSO]). Cells were then stimulated for 15 min with TNF-α or control medium. HA-p105 was isolated from cell lysates by immunoprecipitation, resolved by SDS-6% PAGE, and then Western blotted with anti-p105C antibody. Ip, immunoprecipitates; WT, wild type. (B) The indicated HeLa clones were pretreated with LLnL inhibitor and stimulated with TNF-α as described for that in panel A. Anti-HA immunoprecipitates were Western blotted with anti-p105C antibody.

FIG. 6.

FIG. 6.

βTrCP is required for TNF-α-induced p105 ubiquitination and proteolysis. (A) HeLa cells stably transfected with HA-p105 were transiently transfected with vectors encoding FL-βTrCP1 and FL-βTrCP2. After 48-h culture, cells were stimulated for 15 min with TNF-α or left unstimulated. Anti-HA immunoprecipitates (Ip) were Western blotted with the indicated antibodies. FL-βTrCP expression in cell lysates was confirmed by anti-FL MAb immunoprecipitation and Western blotting. WT, wild type. (B) HeLa cells were pretreated with βTrCP siRNAs complementary to both βTrCP1 and βTrCP2 (−βTrCP) or with control buffer (+βTrCP). After 72 h, cells were stimulated with TNF-α (15 min) or left unstimulated and then lysed. In the upper gel, βTrCP expression of unstimulated cells was determined by Western blotting of phospho-IκBα peptide pulldowns. In the lower gel, cell lysates were Western blotted for IκBα. (C) HeLa cells stably expressing HA-p105 were pretreated as described for panel B. Turnover of HA-p105 in TNF-α-stimulated and control unstimulated cells was determined by pulse-chase metabolic labeling as done for that shown in Fig. 2. Similar results were obtained on two other occasions. (D) βTrCP expression in HA-p105 HeLa cells was suppressed by siRNA pretreatment as described for panel B. HA-p105 ubiquitination induced by 15-min TNF-α stimulation was determined as described in the legend to Fig. 5. (E) βTrCP expression was decreased by siRNA treatment as described for panel B. Phosphorylation of endogenous p105 after TNF-α treatment (15 min) was determined by immunoprecipitation and Western blotting with the indicated antibodies.

FIG. 7.

FIG. 7.

Efficient binding of βTrCP to p105 requires phosphorylation of p105 serines 927 and 932. (A) GST-p105758-967 fusion protein, phosphorylated by His6-IKK2 or left unphosphorylated, was coupled to glutathione-Sepharose beads and used to affinity purify βTrCP translated and labeled with [35S]methionine in vitro. The indicated peptides were added to a final concentration of 100 μg/ml during the pulldown. Isolated βTrCP was detected by autoradiography of SDS-8% PAGE gels. (B) The indicated peptides were coupled to Affi-Gel 10 beads and used as affinity matrices to isolate from HeLa cell lysates endogenous βTrCP, which was detected by Western blotting. (C and D) IκBα, translated and labeled in vitro with [35S]methionine, was first phosphorylated with recombinant IKK2-EE. Labeled protein was then ubiquitinated in vitro (26) in the presence of the indicated peptides or with no added peptide (w/o peptide). Ubiquitination of unphosphorylated IκBα (w/o IKK) is shown as a negative control. IκBα ubiquitination was revealed by autoradiography of SDS-8% PAGE gels. (E) The extent of IκBα ubiquitination in vitro (carried out as described for panel C) was quantified on a phosphorimager over a range of competing peptide concentrations.

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