N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome

Abstract

Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH(2) terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized.

FIG. 1.

A lysineless mutant of ERK3 is unstable. (A) Schematic representation of ERK3Δ constructs showing the position of lysines mutated in this study. (B) HEK 293 cells were transfected with the indicated constructs. After 48 h, cell lysates were analyzed by immunoblotting with anti-HA antibody. Two different exposures of the gel are shown (upper two panels). The same constructs were translated in vitro in the presence of ³⁵S-labeled amino acids and were analyzed by fluorography (lower panel). (C) HEK 293 cells transfected with the different ERK3 constructs were treated with cycloheximide (100 μg/ml) for the indicated times. Ectopically expressed ERK3 was detected by HA immunoblotting. (D) HEK 293 cells were transfected with Myc₆-tagged ERK3Δ, ERK3Δ kinase dead (KD), ERK3Δ Ser189Ala (S189A), ERK3Δ-0K, or ERK3Δ_1-296. The cells were metabolically labeled with ³²P, and the transfected ERK3 proteins were immunoprecipitated with anti-Myc antibody. Phosphorylation was revealed by autoradiography (upper panel). Aliquots of cell lysates were analyzed by immunoblotting to monitor expression of the ectopic proteins (lower panel). exp., exposure; IB, immunoblot; IP, immunoprecipitation.

FIG. 2.

ERK3 is degraded by the proteasome in a lysine-independent manner. (A) HEK 293 cells were transfected with HA-tagged ERK3Δ or ERK3Δ-0K constructs. After 36 h, the cells were treated for 12 h with the protease inhibitors MG-132 (25 μM), E-64 (25 μM), lactacystin β-lactone (20 μM), or vehicle (0.1% dimethyl sulfoxide [DMSO]). Ectopically expressed ERK3 was detected by HA immunoblotting. (B) HEK 293 cells were transfected as described for panel A. After 44 h, the cells were pretreated with dimethyl sulfoxide or MG-132 for 30 min. Cycloheximide was then added for a period of 4 h. Cell lysates were analyzed by HA immunoblotting. IB, immunoblot.

FIG. 3.

Lysineless ERK3 is ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its efficient degradation. (A) Parental BALB/c 3T3 (A31) and E1-thermosensitive mutant (ts20) cells were transfected with the indicated ERK3 constructs or empty vector (V). After 24 h, the cells were maintained at the permissive temperature (34°C) or shifted to the restrictive temperature (39°C) for the indicated times. Ectopically expressed ERK3 was detected by HA immunoblotting. (B) HEK 293 cells were cotransfected with expression vectors encoding ERK1, ERK3Δ, or ERK3Δ-0K together with HA-tagged ubiquitin. The protein kinase constructs contain an N-terminal HA tag and a C-terminal His₆ affinity tag. After 36 h, the cells were treated with MG-132 for 12 h. His₆-tagged proteins were purified from cell lysates with nickel-agarose beads and were analyzed by immunoblotting with anti-HA antibody (upper panel). Asterisks mark nonspecific bands. Total cell lysates were analyzed for global ubiquitination activity by HA immunoblotting (lower panel). IB, immunoblot.

FIG. 4.

Addition of large N-terminal tags stabilizes ERK3 expression. (A) HEK 293 cells were transfected with the indicated constructs. After 48 h, endogenous and ectopic ERK3 were detected by immunoblotting with antibody E3-CTD4, which specifically recognizes the ERK3 C terminus (upper panel). The asterisk marks a nonspecific band. The same constructs were translated in vitro in the presence of ³⁵S-labeled amino acids and were analyzed by fluorography (lower panel). (B) HEK 293 cells were transfected with ERK3 constructs or empty vector (V). After 48 h, the cells were treated with cycloheximide for the indicated times. His₆-ERK3 was purified from cell lysates with nickel-agarose beads and detected by immunoblotting with antibody E3-CTD4 (upper panel). EGFP-ERK3 was analyzed by immunoblotting using anti-GFP antibody (lower panel). (C) HEK 293 cells were transfected as described for panel B and were treated with MG-132 (25 μM) for 12 h. (D) N-terminal tags larger than ∼5 kDa enhance ERK3 expression. HEK 293 cells were transfected with ERK3 constructs tagged at their N termini with an increasing number of copies of the Myc epitope. After 48 h, endogenous and transfected ERK3 was detected by immunoblotting with E3-CTD4 antibody (upper panel). The same constructs were in vitro translated and detected by fluorography (lower panel). (E) The half-lives of ectopically expressed Myc₁-ERK3 (upper panel) and Myc₃-ERK3 (lower panel) were evaluated by cycloheximide chase. (F) The half-lives of Myc₆-ERK3Δ (upper panel) and Myc₆-ERK3Δ-0K (lower panel) were evaluated as described for panel E. IB, immunoblot; IP, immunoprecipitation.

FIG. 5.

The presence of a large N-terminal tag inhibits ERK3 ubiquitination. (A) HEK 293 cells were transfected with ERK3 constructs fused to the EGFP sequence at the N-terminal or C-terminal extremity. The transfected proteins were detected by immunoblotting with anti-GFP antibody (upper panel). The same constructs were in vitro translated and detected by fluorography (lower panel). (B) HEK 293 cells were cotransfected with the indicated ERK3-EGFP fusion constructs together with HA-ubiquitin. The ERK3 constructs contain the GST sequence at the C terminus to allow for specific and quantitative recovery of the transfected proteins. After 36 h, the cells were treated with MG-132 for 12 h. Transfected proteins were purified from cell lysates with glutathione-agarose beads, and comparable amounts of ERK3-EGFP fusion proteins were analyzed. Ubiquitin conjugates were detected by anti-HA immunoblotting (upper panel). The membrane was reprobed with anti-EGFP antibody to confirm that an equivalent amount of ERK3 fusion proteins was loaded on the gel (middle panel). Total cell lysates were analyzed for global ubiquitination activity by HA immunoblotting (lower panel). IB, immunoblot.

FIG. 6.

Addition of large N-terminal tags stabilizes expression of ERK3 and p21 but not that of proteins ubiquitinated on internal lysine residues. (A) Schematic representation of the constructs used in these experiments. Note that proteins expressed from these two vectors are tagged with a single HA epitope. (B to E) HEK 293 cells were transfected with empty vector or with either HA- or Myc₅-HA (M₅HA)-tagged expression vectors encoding ERK1 (B), ERK3 (C), SOCS3 (D), and p53 (E). After 48 h, the HA-tagged proteins were detected by immunoblotting with anti-HA antibody (upper panel). An asterisk denotes a nonspecific band. The same constructs were translated in vitro (IVT) and were analyzed by anti-HA immunoblotting (lower panel). (F) HEK 293 cells were transfected with the same vectors encoding IκBα. After 24 h, the cells were serum starved for 24 h and then left untreated or treated with TNF-α (50 ng/ml) for 30 min in the presence of cycloheximide. HA-tagged proteins were detected as described for panel B. (G) HEK 293 cells were transfected with HA- or M₅HA-tagged p21. After 48 h, the expression of ectopic p21 was monitored by HA immunoblotting. (H) HEK 293 cells transfected with p21 expression vectors were treated with cycloheximide for the indicated times. HA-tagged proteins were detected as described for panel B. ORF, open reading frame; IB, immunoblot.

FIG. 7.

Degradation of lysineless p21 is dependent on a functional ubiquitin conjugation pathway. (A) Parental A31 and E1-mutant ts20 cells were cultured at 34 or 39°C for the indicated times. Expression of endogenous p21 was monitored by immunoblotting with anti-p21 antibody. (B) Half-life of endogenous p21 was measured by cycloheximide chase experiments in ts20 cells grown for 12 h at the permissive or restrictive temperature. (C) A31 or ts20 cells were transfected with the indicated p21 constructs or empty vector (V). The p21 constructs contain an N-terminal HA tag and a C-terminal His₆ tag. After 24 h, the cells were maintained at 34 or 39°C for the indicated times. Ectopically expressed p21 proteins were purified from cell lysates with nickel-agarose beads and were analyzed by immunoblotting with anti-HA antibody. (D) HEK 293 cells were cotransfected with the indicated p21 expression vectors together with HA-tagged ubiquitin. After 36 h, the cells were treated with MG-132 for 12 h. His₆-tagged proteins were purified from cell lysates with nickel-agarose beads. Half of the purified material was separated on a 7.5% gel (upper panel), while the other half was loaded on a 12% gel (middle panel). Ubiquitin conjugates were detected by HA immunoblotting. The arrow indicates the position of the nonubiquitinated p21 protein. An asterisk marks a nonspecific band. Total cell lysates were analyzed for global ubiquitination activity by HA immunoblotting (lower panel). IB, immunoblot.

FIG. 8.

Identification of N-terminally ubiquitinated ERK3 and p21 by MS-MS. (A) Schematic representation of the constructs used in this study. The ERK3Δ and p21 fusion proteins are tagged with an N-terminal HA peptide, which upon tryptic digestion releases the sequence MYDVPDYASLPGNYR (boldface characters identify the HA peptide) together with an extra diglycine derived from the C terminus of ubiquitin. (B) HEK 293 cells were transfected with the indicated constructs and treated for 12 h with MG-132. The transfected proteins were purified from cell lysates with glutathione-agarose beads and were analyzed by LC-MS-MS. The fragmentation pattern of the ubiquitinated N-terminal HA peptide is shown. The tandem mass spectrum of the doubly protonated precursor ion at m/z 928.8 shows a series of y-type fragment ions arising from peptide cleavage with charge retention at the C terminus, thus confirming the expected HA peptide sequence containing the methionine residue. The cyclization of the amine of the terminal glycine to the neighboring aspartate residue is supported by a characteristic b5 fragment ion at m/z 506.1, corresponding to the cleavage of the amide bond with charge retention at the N terminus.