Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin-3

Abstract

Deubiquitinating enzymes (DUBs) control the ubiquitination status of proteins in various cellular pathways. Regulation of the activity of DUBs, which is critically important to cellular homoeostasis, can be achieved at the level of gene expression, protein complex formation, or degradation. Here, we report that ubiquitination also directly regulates the activity of a DUB, ataxin-3, a polyglutamine disease protein implicated in protein quality control pathways. Ubiquitination enhances ubiquitin (Ub) chain cleavage by ataxin-3, but does not alter its preference for K63-linked Ub chains. In cells, ubiquitination of endogenous ataxin-3 increases when the proteasome is inhibited, when excess Ub is present, or when the unfolded protein response is induced, suggesting that the cellular functions of ataxin-3 in protein quality control are modulated through ubiquitination. Ataxin-3 is the first reported DUB in which ubiquitination directly regulates catalytic activity. We propose a new function for protein ubiquitination in regulating the activity of certain DUBs and perhaps other enzymes.

Figure 1

Endogenous AT3 is ubiquitinated under basal conditions, and its ubiquitination is enhanced during proteasome inhibition. (A) Schematic of AT3 showing the N-terminal catalytic (Josephin) domain and the C-terminal Ub-interacting motifs (UIMs) flanking the polyglutamine tract. (B) AT3 is ubiquitinated in cells. FLAG–AT3 and HA–Ub were coexpressed in Cos7 cells. Lysates were subjected to stringent immunopurification with anti-FLAG antibody, then probed with anti-AT3 and anti-HA antibodies. Several high molecular weight ubiquitinated AT3 (AT3-Ub) bands are recognized by both anti-AT3 (1H9) and anti-HA antibodies. (C) Endogenous AT3 is ubiquitinated. Left: M17 cells were treated with or without the proteasome inhibitor lactacystin. Several higher molecular weight AT3-Ub species present in untreated cells are enriched when the proteasome is inhibited. Right: AT3 immunoprecipitated with polyclonal anti-AT3 antibody (MJD) from cells treated with lactacystin was probed with monoclonal anti-AT3 (1H9) and anti-Ub (P4D1) antibodies, confirming that the higher molecular weight forms of AT3 are ubiquitinated. Asterisks: mono-ubiquitinated AT3 exists in cells under basal conditions. AT3 doublets probably reflect allelic differences in the CAG/polyQ repeat in M17 cells. Ctrl: polyclonal, anti-HA antibody.

Figure 2

Ubiquitinated AT3 shows enhanced catalytic activity. (A) Diagram of preparation and immunoprecipitation of unmodified and ubiquitinated FLAG–AT3 from Cos7 cells transfected as indicated. (B) Ubiquitinated AT3 immunopurified from cells cleaves K63-Ub6 chains more rapidly than does unmodified AT3. AT3(WT), AT3(WT)-Ub, or catalytically inactive AT3-Ub (AT3(C14A)-Ub) (50 nM) were incubated with K63-Ub6 chains (250 nM). HMW: high molecular weight Ub species. Bottom: membrane was stripped and probed with anti-AT3 antibody. (C) AT3 ubiquitinated in vitro cleaves K63-Ub6 chains more quickly than does unmodified AT3. Anti-AT3 blot shows GST–AT3 species used in reactions.

Figure 3

Accelerated cleavage of K63-Ub6 chains by AT3-Ub. Left: untagged AT3 or AT3-Ub (ubiquitinated in vitro) was incubated with K63-Ub6 chains for the indicated times. Anti-AT3 blot shows AT3 species used in reactions. Right: time-course of Ub reaction product appearance, determined by semi-quantification of western blots as shown on the left. Means±standard deviations (s.d.), N=5. Asterisks: statistically significant difference at P<0.001.

Figure 4

The UIMs of AT3 are not necessary for activation by ubiquitination. (A) Ubiquitinated, UIM-mutated AT3 shows enhanced activity towards K63-Ub6 chains. Left: wild-type (AT3(WT)) or UIM-deficient AT3 (AT3(UIM^*)) was ubiquitinated in vitro, then incubated with K63-Ub6 chains. Right: semi-quantitative representation of western blot results as on the left. Means±s.d.; N=6. (B) AT3(UIM^*)-Ub cleaves K48-Ub5 chains more quickly than AT3(UIM^*). Left: AT3 species were incubated with K48-Ub5 chains. Right: semi-quantitative representation of four independent experiments. Means±s.d. Anti-AT3 blots show GST–AT3 species used in reactions in (A) and (B).

Figure 5

Ubiquitination of the Josephin domain enhances cleavage of K48-linked Ub chains. (A) Ubiquitination of the Josephin domain does not enhance activity towards K63-Ub6 chains. Left: Josephin domain of AT3 (AT3(J)) was ubiquitinated in vitro and incubated with K63-Ub6 chains. Right: semi-quantitative representation of experiments conducted as on the left. Means±s.d.; N=3. (B) Left: ubiquitinated Josephin domain has enhanced activity towards K48-Ub5 chains. Right: semi-quantitative representation of experiments conducted as on the left. Means±s.d.; N=5. Asterisks: statistically significant difference at P<0.01. GST-tagged and untagged Josephin domain proteins yield similar results. (C) AT3 activation by ubiquitination does not occur in trans. AT3 species were prepared in vitro. Catalytically inactive AT3 (AT3(C14A)), either unmodified or ubiquitinated, was incubated with wild-type AT3 (AT3(WT)) and K63-Ub6 chains. Anti-AT3 blot shows GST–AT3 species used in reactions. Results representative of three independent experiments are shown.

Figure 6

Expanded (pathogenic) AT3 is also activated by ubiquitination. (A) Recombinant, expanded AT3 (AT3(Q80)) was ubiquitinated in vitro, then incubated with the indicated Ub chains. AT3(Q80)-Ub shows greater enhancement of activity towards K63-Ub6 chains. (B) Normal and expanded AT3 show similar enhancement in activity when ubiquitinated. Results in (A) and (B) are each representative of three independent experiments. Anti-AT3 blots show GST–AT3 species used in reactions.

Figure 7

Levels of ubiquitinated endogenous AT3 are enhanced by certain stressors. (A) The fraction of endogenous AT3 ubiquitinated under basal conditions is enhanced by proteasome inhibition or by increasing Ub levels. Left: top blot shows Cos7 cells treated with lactacystin (10 μM; 10 h), or transfected with HA–Ub and treated as indicated. Bottom blot shows the same cell lysates loaded in 4–20% SDS–PAGE gel to probe for conjugated and non-conjugated Ub. All lanes are from the same exposure of the same blot. Right: AT3 was immunoprecipitated from Cos7 cells treated with lactacystin. Endogenous AT3-Ub bands are detected by both anti-AT3 and anti-Ub antibodies, confirming the identity of HMW AT3 species on the left as AT3-Ub. Representative results from four independent experiments are shown. (B) Induction of the unfolded protein response transiently leads to higher levels of ubiquitinated endogenous AT3. Treatment of Cos7 or 293 cells with the UPR inducer, DTT (5 μM), for the indicated times leads to the appearance of a higher molecular AT3 band consistent with ubiquitinated AT3. AT3 doublets in 293 cells most likely reflect allelic differences in CAG/polyQ repeat length. Representative results from at least three independent experiments are shown. (C) Heat shock does not alter the levels of ubiquitinated endogenous AT3. Cos7 cells were heat-shocked briefly (left) or for a prolonged time (right). Hsp70 levels confirmed induction of the heat shock response in treated cells. Representative results from at least three independent experiments are shown. Equal protein was loaded in (A–C). (D) Pathogenic AT3 is more heavily ubiquitinated than unexpanded AT3 in brain lysates from transgenic mice expressing normal (AT3(Q15)) or expanded (AT3(Q84)) AT3. Left: AT3(Q84) is more heavily ubiquitinated than AT3(Q15). Asterisk: endogenous AT3. AT3(Q15) mice express the protein more highly than the AT3(Q84) mice. Right: stringent immunopurification of 12-month-old AT3(Q84)-expressing brains shows that the HMW bands in left (AT3(Q84)-Ub) are ubiquitinated AT3.

Figure 8

Effects of protein domains on catalytic properties of AT3. Ubiquitination of the Josephin domain is sufficient for activation. Although the UIMs are not necessary for AT3 activation by ubiquitination, they confer Ub linkage preference to the catalytic domain.