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. 2017 Apr 4;114(14):3565-3571.
doi: 10.1073/pnas.1700949114. Epub 2017 Mar 20.

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Affiliations

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Thang Van Nguyen et al. Proc Natl Acad Sci U S A. .

Abstract

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4CRBN and p97 pathways.

Keywords: CRBN; VCP/p97; degradation; glutamine synthetase; substrates.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Glutamine alters the apparent assembly state of GS in an ubiquitylation- and p97-dependent manner. (A and B) MCF7 cells were starved of glutamine for 24 h and then were pretreated with (A) or without (B) 2 µM pan cullin–RING ubiquitin ligase inhibitor MLN4924 for 30 min, followed by the addition of 4 mM glutamine plus the histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) (2 µM) and NAM (10 µM) (+Q +HDACi) or not (−Q −HDACi) for 2 h. Cell lysates were fractionated on a Superdex 200 gel filtration column. Individual fractions were concentrated by trichloroacetic acid (TCA) precipitation and analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. (C) As in A and B, except that H1299 cells were starved of glutamine for 24 h and then were pretreated with MLN4924 (2 μM) or the GS inhibitor MSO (2 mM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. (D) As in A and B, except that HEK293 cells were starved of glutamine for 24 h and then were pretreated with MLN4924 (2 μM) or the p97 inhibitor CB-5083 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. (E) Fractions 10–12, prepared from HEK293 cells treated with CB-5083 and glutamine (used in D, bottom panel), were combined and subjected to pulldown with TUBE2 resin, followed by treatment with or without USP2. The bound fractions were analyzed by SDS/PAGE and immunoblotting with antibodies against GS and ubiquitin. L.E., long exposure; S.E., short exposure; (Ub)n, polyubiquitin.
Fig. S1.
Fig. S1.
(Related to Fig. 1) The GS inhibitor MSO suppresses glutamine-induced GS ubiquitylation and degradation. (A) H1299 and MCF7 cells were starved of glutamine for 24 h. Cells were pretreated with the GS inhibitor MSO for 30 min, followed by the addition (or not) of 4 mM glutamine for 8 h. Cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, CRBN, and GAPDH. (B and C) H1299 (B) and MCF7 (C) cells were starved of glutamine for 24 h and then were pretreated with MG132 (10 μM) in the presence or absence of MSO (1 mM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 3 h. Cell lysates were fractionated on a TUBE2 resin. The bound fractions and lysate samples (inputs) were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin, and GAPDH. (Ub)n, polyubiquitin.
Fig. 2.
Fig. 2.
p97 interacts with endogenous GS and promotes glutamine-induced degradation of GS. (A) HEK293 cells were starved of glutamine for 24 h, were pretreated (or not) with MLN4924 (2 μM) for 30 min, and then were treated (or not) with 4 mM glutamine for 2 h. For CB-5083 treatment, after the addition of 4 mM glutamine for 90 min, cells were treated with CB-5083 (10 μM) for 30 min. Protein extracts were immunoprecipitated with mouse IgG control or p97 antibodies, followed by Western blot analysis with the indicated antibodies. The ratio of GS bound to p97 normalized to input GS is shown. L.E., long exposure (the long exposure blot was quantified); S.E., short exposure; (Ub)n, polyubiquitin. (B and C) MCF7 cells were starved of glutamine for 24 h and were pretreated with the p97 inhibitors CB-5083 (B) or NMS-873 (C) for 30 min, followed by addition (or not) of 4 mM glutamine for 4 h. Cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, p97, and GAPDH. The relative ratios of GS:GAPDH, normalized to lane 1, are shown. (D) HEK293 cells stably expressing doxycycline (Dox)-inducible shRNA targeting p97 were either mock-treated or induced with doxycycline (1 µg/mL) for 48 h and then were starved of glutamine for 24 h, followed by the addition of 4 mM glutamine for the indicated times. Cell lysates were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, p97, and GAPDH. The relative ratios of GS:GAPDH, normalized to lane 1, are shown. (E) H1299 cells were starved of glutamine for 24 h and then were pretreated with MG132 (10 μM) or CB-5083 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 3 h. Cell lysates were fractionated on a TUBE2 resin, followed by treatment with or without USP2. The bound fractions and lysate samples were analyzed by SDS/PAGE and immunoblotting with antibodies against GS and ubiquitin. Input is shown in Fig. S3B. (F) HEK293 cells stably expressing doxycycline-inducible shRNA targeting p97 were mock-treated or were induced with doxycycline (1 µg/mL) for 48 h and then were starved of glutamine for 24 h, followed by the addition of 4 mM glutamine for the indicated times. Cell lysates were fractionated on a TUBE2 resin, and both lysate samples and the bound fractions were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin (shown in Fig. S3C), p97, and GAPDH.
Fig. S2.
Fig. S2.
(Related to Fig. 2) p97 interacts with endogenous GS and is required for glutamine-induced GS degradation. (A) HEK293 cells were starved of glutamine for 24 h and were pretreated (or not) with CB-5083 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. Protein extracts were immunoprecipitated with mouse IgG control or p97 antibodies, followed by Western blot analysis with the indicated antibodies. The ratios of GS bound to p97 normalized to input GS are shown. (Ub)n, polyubiquitin. (B) HEK293 cells were starved of glutamine for 24 h and were pretreated with the p97 inhibitor NMS-873 or CB-5083 for 30 min, followed by the addition (or not) of 4 mM glutamine for 4 h. Cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, p97, and GAPDH. The relative ratio of GS:GAPDH, normalized to lane 1, is shown. (C) HEK293 cells stably expressing doxycycline (Dox)-inducible shRNA targeting p97 were mock-treated or were induced with doxycycline (1 µg/mL) for 48 h and then were starved of glutamine for 24 h, followed by the addition of 4 mM glutamine and the HDAC inhibitors (2 μM SAHA+10 mM NAM) for the indicated times. Cell extracts were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, p97, and GAPDH. The GS:GAPDH ratio for each sample was calculated, normalized to untreated cells, and is indicated below the bottom immunoblot in each panel.
Fig. S3.
Fig. S3.
(Related to Fig. 2) p97 controls GS ubiquitylation status in response to glutamine. (A) H1299 cells were starved of glutamine for 24 h and then were pretreated with CB-5083 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 3 h. Cell lysates were fractionated on a TUBE2 resin. The bound fractions and lysate samples were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin, and GAPDH. (Ub)n, polyubiquitin. (B) Western blot analysis showing input protein levels for Fig. 3B. (C) The bound fractions shown in Fig. 2F were analyzed by SDS/PAGE and immunoblotting with anti-ubiquitin antibody. (D) The ATP hydrolysis-deficient p97 E578Q mutant promotes the accumulation of ubiquitylated GS in response to glutamine. H1299 cells were transfected with empty plasmid (EV) or with plasmids expressing WT p97Myc or the p97Myc-E578Q mutant. Twenty-four to thirty-six hours after transfection, cells were starved of glutamine for 24 h, followed by the addition (or not) of 4 mM glutamine for 2 h. Total ubiquitinated proteins were affinity-purified using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. (E) The ATP hydrolysis-deficient p97 E578Q mutant binds endogenous GS. HEK293 cells were transfected with empty plasmid or with plasmids expressing WT p97Myc or the p97Myc-E578Q mutant. Twenty-four hours after transfection, cells were starved of glutamine for 24 h and were pretreated (or not) with CB-5083 (10 μM) for 30 min followed by the addition (or not) of 4 mM glutamine for 2 h. Cell extracts were immunoprecipitated with anti-Myc antibody, and the precipitated and input fractions were evaluated by SDS/PAGE and immunoblotting with the indicated antibodies.
Fig. 3.
Fig. 3.
Inhibition of p97 promotes the accumulation of ubiquitylated GS in a CRL4CRBN-dependent manner. (A) MCF7 cells were starved of glutamine for 24 h and then were pretreated (or not) with CB-5083 (10 μM), NMS-873 (10 μM), or MLN4924 (2 μM) for 30 min as indicated in the figure, followed by the addition (or not) of 4 mM glutamine for 3 h. Cell lysates were fractionated on a TUBE2 resin, and both lysate samples and the bound fractions were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin, and GAPDH. (Ub)n, polyubiquitin. (B) HEK293 cells stably expressing doxycycline (Dox)-inducible shRNA targeting p97 were induced with doxycycline (1 µg/mL) for 48 h and then were starved of glutamine for 24 h. Cells were pretreated (or not) with MLN4924 (2 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. Cell lysates were fractionated on a TUBE2 resin, and both lysate samples and the bound fractions were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin, and GAPDH. (C) CRBN-KO 293FT cells stably expressing WT FlagCRBN or the indicated mutants were starved of glutamine for 24 h. Cells were pretreated with NMS-873 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. Cell lysates were fractionated on a TUBE2 resin, and both lysate samples and the bound fractions were analyzed by SDS/PAGE and immunoblotting with antibodies against GS, ubiquitin, Flag, and GAPDH.
Fig. 4.
Fig. 4.
The p97 adaptor complex UFD1•NPL4 interacts with ubiquitylated GS upon the addition of glutamine. (A) H1299 cells were transfected with control (CT) siRNA or with siRNAs that target NPL4, UFD1, or UBXD7. Forty-eight hours after transfection, cells were starved of glutamine for 24 h, followed by the addition (or not) of 4 mM glutamine for 2 h. Total ubiquitinated proteins were affinity-purified from cell lysates using TUBE2-agarose. Bound fractions and cell lysates (input) were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. (Ub)n, polyubiquitin. (B and C) HEK293 cells were transiently transfected with empty vector or with plasmids encoding FlagNPL4 (B) or UFD1Flag (C). After 24-h transfection, cells were starved of glutamine for 24 h and were pretreated (or not) with MG132 (20 μM), CB-5083 (10 μM), or MLN4924 (2 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 2 h. Cell lysates were immunoprecipitated with anti-Flag resin. Precipitated and input fractions were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies.
Fig. 5.
Fig. 5.
p97 mediates the disassembly of GS upon inhibition of the proteasome. (A) MCF7 cells were grown to 80–90% confluence in 15-cm plates and were starved of glutamine for 24 h. Cells were pretreated (or not) with MG132 (10 μM), NMS-873 (10 µM), or CB-5083 (10 μM) for 30 min and then were treated with 4 mM glutamine (+Q) or not (−Q) for 6 h. Cell lysates were fractionated on a Superdex 200 gel filtration column. Individual fractions were concentrated by TCA precipitation and analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. (B) HEK293 cells were starved of glutamine for 24 h and then were treated with 4 mM glutamine (+Q) or not (−Q) for 2 h. Cell lysates were fractionated on a Superdex 200 gel filtration column. (Upper) Individual fractions were concentrated by TCA precipitation and analyzed by SDS/PAGE and immunoblotting with antibodies against GS and p97. (Lower) Fractions 8–14, prepared from HEK293 cells treated with 4 mM glutamine, were immunoprecipitated with p97 antibody, followed by Western blot analysis with the indicated antibodies. A band at ∼50 kDa represents IgG heavy chains (IgG-HC).
Fig. S4.
Fig. S4.
(Related to Fig. 5) Depletion of p97 by shRNA blocks glutamine-induced GS disassembly. HEK293 cells stably expressing doxycycline (Dox)-inducible shRNA targeting p97 were mock-treated or were induced with doxycycline (1 µg/mL) for 48 h, starved of glutamine for 24 h, and then pretreated with MG132 (10 μM) for 30 min, followed by the addition (or not) of 4 mM glutamine for 6 h. Cell lysates were fractionated on a Superdex 200 gel filtration column. Individual fractions were concentrated by TCA precipitation and analyzed by SDS/PAGE and immunoblotting with the indicated antibodies.
Fig. 6.
Fig. 6.
p97 is required for immunomodulatory drug-induced degradation of CRBN neosubstrates. (A) MM1S cells were pretreated with the indicated doses of CB-5083 or 1 µM MLN4924 (MLN) for 30 min, followed by the addition of lenalidomide (Len) (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. The relative ratios of IKZF1:GAPDH or IKZF3, normalized to lane 1, are shown here and in BD. (B) L363 cells were pretreated with CB-5083 (2 μM) for 30 min, followed by the addition (or not) of lenalidomide (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. (C) MM1S cells stably expressing control (CT) or CRBN shRNAs were pretreated with CB-5083 (2 μM) for 30 min, followed by the addition (or not) of lenalidomide (10 μM) for 5 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (D) As in C, except that cells were pretreated (or not) with NMS-873 (2 or 5 μM) for 30 min. (E) U937 and MM1S cells were pretreated (or not) with CB-5083 (2 or 10 μM), MLN4924 (1 μM), or MG132 (10 μM) for 1 h, followed by treatment with 10 nM CC-885 for an additional 2 h. Whole-cell extracts were subjected to immunoblot analysis. (F) Cells were pretreated with CB-5083 (2 μM) and/or lenalidomide (10 μM) for 30 min followed by the addition of cyclohexamide (CHX) (100 μg/mL). At the indicated times after the addition of cyclohexamide, samples were harvested for immunoblot analysis. The relative ratios of CK1α:GAPDH, normalized to lane 1, are shown.
Fig. S5.
Fig. S5.
(Related to Fig. 6) (A) Lenalidomide-induced degradation of CRBN neosubstrates requires p97. U266 cells were pretreated with the indicated doses of CB-5083 or NMS-873 for 30 min, followed by the addition (or not) of lenalidomide (Len) (10 μM) for 4 h. Cell lysates were fractionated by SDS/PAGE and immunoblotted for the indicated endogenous proteins. (B) Depletion of p97 or proteasome activity promotes the accumulation of ubiquitylated IKZF1, suggesting that p97 functions downstream of IKZF1 ubiquitylation. HEK293 cells were transfected with plasmids encoding HAUb and IKZF1Flag. After 24 h of transfection, the cells were pretreated with 20 µM MG132 or 10 µM CB-5083 for 30 min followed by the addition (or not) of 10 µM lenalidomide for 3 h. Denatured lysate proteins were immunoprecipitated with anti-Flag antibody. The input lysates and bound fractions were evaluated by SDS/PAGE and immunoblotting with antibodies against the HA and Flag tags.
Fig. 7.
Fig. 7.
Proposed model for the role of p97 in mediating regulated degradation of CRL4CRBN substrates, including GS and lenalidomide-induced neosubstrates. For the sake of simplicity, one subunit of GS homodecamer is ubiquitylated by CRL4CRBN, segregated by the p97–UFD1–NPL4 complex, and subsequently degraded by the proteasome upon the addition of glutamine. Ub, ubiquitin.
Fig. S6.
Fig. S6.
(Related to Fig. 3) CRBN expression covaries significantly with p97 and its adaptors. Transcript expression values (expressed as TPM) were obtained from RNA-seq performed on cancer cell lines in the 935 CCLE. These data were generated by the CTD2 network (https://ctd2.nci.nih.gov/dataPortal/) established by the National Cancer Institute’s Office of Cancer Genomics. For each detected transcript, significance of correlation with Crbn across 935 samples was determined. Data are plotted in terms of increasing significance for transcripts positively and negatively correlated with CRBN. p97 and its adaptors, as a whole, are significantly correlated with CRBN mRNA expression as determined by Kolmogorov–Smirnov testing.
Fig. S7.
Fig. S7.
(A) Void volume determination of Superdex 200 10/300 GL. Blue dextran (400 μL, 1 mg/mL) was injected into a Superdex 200 10/300 GL column and was monitored at wavelengths of 280, 380, and 620 nm (A280, absorbance at 280 nm; A380, absorbance at 380 nm; A620, absorbance at 620 nm). (B) Chromatogram for protein standard of Superdex 200 10/300 GL. Gel filtration standard (500 μL) (Bio-Rad; 151–1901) was injected into a Superdex 200 10/300 GL column and was monitored at a wavelength of 280 nm. mAU, milli absorbance unit.

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