The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner

Abstract

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.

Figure 1

Processing of p105(1–971) and GST-p105(365–971) to p50 or p50-like product in vitro by the 20S proteasome. (A) Domain organization of mouse p105 showing NTD, DimD, ARD, and death domain (DD). Positions of NLS, GRR, and phosphorylation sites (P) are indicated. (B) Processing of His-tagged full-length p105(1–971) by 20S proteasome. Reaction products were separated by SDS–PAGE and visualized by Western blotting with p50(NLS) antibody. (C) Processing of GST-tagged p105(365–971) by the 20S proteasome. Reaction products were separated by SDS–PAGE and visualized by Western blotting with GST antibody (left panel) or CTp105 antibody (right panel). (D) Proteasome activity assays to test activity of 20S proteasome toward a fluorogenic peptide substrate. The activity with and without 0.03% SDS is indicated by solid and dotted lines, respectively.

Figure 2

p50 generation in vivo is independent of translation and requires ankyrin repeat containing precursors for precise processing. (A) HEK293T cells transfected (right panel) or untransfected (left panel) with Flag-tagged full-length p105 were pulse-radiolabeled with ³⁵S-Met for 30 min and chased for the indicated time. Cell lysates were immunoprecipitated with Flag antibody and separated by SDS–PAGE and visualized by fluorography. The schematic diagram for p105 is given as a residue numbering guideline. (B) HEK293T cells transfected with various YFP-tagged p105 truncations or with the empty vector. The precursors and processed products were separated on 10% (15% in insets) SDS–PAGE gel and visualized by Western blotting with GFP antibody. The schematic diagram for p105 is given as a residue numbering guideline. (C) HEK293T cells transfected with various YFP-tagged p105 constructs, and analyzed at indicated times after transfection. Levels of precursor and product proteins were assessed by Western blotting with GFP antibody. The schematic representation of constructs used is given.

Figure 3

p50 generation in vivo is Ub-independent. (A) Temperature-sensitive E1 mutant ts20 cells maintained at the permissive (P) or restrictive (R) temperatures. Levels of p50 and p105 in lysates from cells grown in P and R temperatures were assessed by Western blotting with p50(NLS) antibody (top panel). Ub-dependent cellular p53 accumulated at the R temperature in ts20 cells (bottom panel). (B) HEK293T cells were transfected with expression plasmids encoding wild-type p105 (WT) and lysine to arginine mutant (KtoR) of p105 fused to YFP. The minimal processing labile p105, 1–465 and 245–465, were used. Proteins were separated on 15% (left panel) and 10% (right panel) SDS–PAGE gel and visualized by Western blotting with GFP antibody. The schematic diagram for the p105 constructs with the positions of the mutated lysines is given.

Figure 4

p105 processing is an endoproteolytic event. (A) 20S proteasome endoproteolytically cleaves GST-p105(365–971)-GFP in vitro as detected by generation of GST-related products by Western blotting with GST antibody. (B, C) 20S proteasome endoproteolytically cleaves GST-p105(365–971)-GFP in vitro as detected by generation of GFP-related products by Western blotting with GFP antibody. GST-GFP, GST, and GFP remain stable over the course of the reaction. The schematic diagram of proteins used is given. (D) GFP fluorescence (excitation 395 nm, emission 508 nm) of GST-p105(365–971)-GFP and GST-GFP was monitored during the 20S proteasomal reaction. (E) HEK293T cells transfected with various YFP-p105-GFP double fusion proteins as indicated schematically on top. Lane 9 represents the empty GFP vector control transfection. The precursors and processed products were visualized by Western blotting with p50(NLS) antibody.

Figure 5

GRR protects upstream domains from degradation by the 20S proteasome. (A) The left panel shows that in vitro 20S proteasome generates stable products from GST-p105(365–971) and GST-p105(365–800), but not from GST-p105(435–971), which lacks the GRR. The size of the product generated is equivalent to the size of recombinant GST-GRR. The right panel shows that the GST-GRR generated from GST-p105(365–971) is stable over a prolonged period of incubation. Precursors and products were visualized by Western blotting with GST antibodies. The schematic diagram of proteins used is given. (B) Stable p50-like product, His-p50(∼1–430), is generated from the full-length p105, but not from mutant p105, which lacks GRR, p105Δ(356–498) (left and middle panels). RHR, p105(39–363) was stable over the same period of incubation with 20S proteasome (right panel). Proteins were visualized by both Coomassie staining and Western blotting with p50 antibody. The schematic diagram of proteins used in the in vitro 20S proteasome assay is given on top. (C) HEK293T cells were transfected with full-length p105 or p105 deletion mutant p105Δ(356–498) as HA (left panel) or YFP (right panel) fusion proteins. Cell extracts were separated by SDS–PAGE followed by Western blotting with HA and GFP antibodies. ns refers to nonspecific band. (D) HEK293T cells transfected (right and middle panels) or untransfected (left panel) with Flag-tagged full-length p105 and p105Δ(356–498) were pulse-radiolabeled with ³⁵S-Met for 30 min and chased for the indicated time. Cell lysates were immunoprecipitated with Flag antibody and separated by SDS–PAGE and visualized by fluorography.

Figure 6

Model for p105 processing. In vitro studies have shown that p105 exists as a dimer. Folding back of the C-terminal of p105 masks the NLS and presents the correct processing region for cleavage by the 20S proteasome. Upon cleavage, the C-terminal is preferentially degraded, while the GRR protects the N-terminal from further degradation, thereby generating p50.