Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability

Abstract

Melanoma antigen genes (MAGEs) are emerging as important oncogenic drivers that are normally restricted to expression in male germ cells but are aberrantly expressed in cancers and promote tumorigenesis. Mechanistically, MAGEs function as substrate specifying subunits of E3 ubiquitin ligases. Thus, the activation of germline-specific genes in cancer can drive metabolic and signaling pathways through altered ubiquitination to promote tumorigenesis. However, the mechanisms regulating MAGE expression and activity are unclear. Here, we describe how the MAGE-A3/6 proteins that function as repressors of autophagy are downregulated in response to nutrient deprivation. Short-term cellular starvation promotes rapid MAGE-A3/6 degradation in a proteasome-dependent manner. Proteomic analysis reveals that degradation of MAGE-A3/6 is controlled by the CRL4-DCAF12 E3 ubiquitin ligase. Importantly, the degradation of MAGE-A3/6 by CRL4-DCAF12 is required for starvation-induced autophagy. These findings suggest that oncogenic MAGEs can be dynamically controlled in response to stress to allow cellular adaptation, autophagy regulation, and tumor growth and that CRL4-DCAF12 activity is responsive to nutrient status.

Keywords: MAGE; DCAF12; starvation; ubiquitin.

Figure 1. Degradation of MAGE‐A3/6 proteins upon nutrient deprivation

1. A, B

   MAGE‐A3/6 protein levels decrease upon EBSS treatment of HeLa cells for the indicated times. Note both bands are MAGE‐A3/6. Quantitation represents average of n = 3 with standard deviation shown.

2. C

   Quantitative TMT proteomics of A375 cells grown in complete media (CM) or EBSS for 3 h. Number of proteins affected are shown. Autophagy‐related proteins are indicated in gray and MAGE‐A proteins in blue.

3. D, E

   MAGE‐A3/6 protein levels are actively degraded upon nutrient deprivation. HeLa cells were treated for the indicated times with complete media (DMEM + 10% FBS) or EBSS containing 100 μg/ml translation inhibitor cycloheximide (CHX). Protein degradation rates are shown (E). Quantitation represents average of n = 3 with standard deviation shown.

Data information: Asterisks indicate **P < 0.01. ns indicates P > 0.05 using two‐way ANOVA.

Figure 2. MAGE‐A3/6 proteins are stabilized by insulin in FBS

1. A, B

   FBS (10%), but not non‐essential amino acids (NEAA; 10 mM) or glucose (4.5 g/l), rescues MAGE‐A3/6 protein levels upon EBSS treatment. HeLa cells were treated with EBSS and the indicated supplements for the indicated times. Quantitation represents average of n = 3 with standard deviation shown.

2. C

   Removal of FBS from normal growth conditions (DMEM + 10% FBS) results in MAGE‐A3/6 protein degradation in HeLa cells.

3. D, E

   MAGE‐A3/6 protein stability is controlled by a < 3 kDa, heat stable, lipophilic molecule present in FBS. HeLa cells were treated with EBSS alone, or EBSS containing 10% complete FBS, 10% charcoal–dextran‐stripped FBS, 10% boiled FBS, 10% dialyzed > 3 kDa FBS, or 10% dialyzed < 3 kDa FBS for the indicated times. Quantitation represents average of n = 3 with standard deviation shown.

4. F

   MAGE‐A3/6 protein instability upon nutrient deprivation of HeLa cells is not altered by EGF 0.2 ng/ml, testosterone, or bovine pituitary extract 25 μg/ml.

5. G, H

   Insulin partially rescues MAGE‐A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times with EBSS alone or EBSS containing NEAA (10 mM) or insulin (10 μg/ml). Quantitation represents average of n = 3 with standard deviation shown.

Data information: Asterisks indicate ***P < 0.001. ns indicates P > 0.05 using two‐way ANOVA.

Figure 3. MAGE‐A3/6 is degraded by the proteasome upon nutrient deprivation

1. A

   MAGE‐A3/6 mRNA levels do not change upon nutrient deprivation. RNA was isolated from HeLa cells treated with EBSS for the indicated times. RT‐qPCR analysis was performed to detect both MAGE‐A3/6 with a common primer set. Data were normalized to 18S rRNA levels. Quantitation represents average of n = 3 with standard deviation shown.

2. B, C

   Proteasome inhibition rescues MAGE‐A3/6 protein levels upon nutrient deprivation. HeLa cells were treated for the indicated times in EBSS alone or EBSS containing lysosome inhibitor bafilomycin A1 (BafA1, 50 nM) or proteasome inhibitor MG132 (10 μM). Quantitation represents average of n = 3 with standard deviation shown.

3. D

   Nutrient deprivation increases ubiquitination of endogenous MAGE‐A3/6. HeLa cells were treated with MG132 (10 μM) in complete media or EBSS for 6 h. Cell extracts were incubated with control agarose or agarose‐TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.

4. E

   MAGE‐A3/6 protein instability upon nutrient deprivation is not rescued by knockdown of its associated E3 ubiquitin ligase, TRIM28. HeLa cells were treated with control or TRIM28 siRNAs for 6 days before incubation in EBSS for the indicated times.

5. F, G

   MAGE‐A3/6 degradation upon nutrient starvation is dependent on a cullin E3 ubiquitin ligase. HeLa cells were treated with EBSS alone or EBSS containing the NAE1 inhibitor MLN4924 (1 μM) that blocks the activity of cullin E3 ubiquitin ligases for the indicated times. Quantitation represents average of n = 3 with standard deviation shown.

6. H

   Degradation of MAGE‐A3/6 upon nutrient deprivation is dependent on a Cul4 E3 ubiquitin ligase complex. HeLa cells were transfected with vector control or the indicated dominant‐negative (D/N) cullin constructs for 48 h before harvesting cells after 0 or 4 h EBSS treatment.

7. I

   Degradation of MAGE‐A3/6 upon nutrient deprivation is dependent on Cul4A/B. HeLa cells were transfected with control siRNAs or siRNAs targeting both Cul4A and Cul4B. 72 h after treatment, cells were incubated for the indicated times with EBSS before harvesting and immunoblotting. Note, time 0 samples between two siRNA groups were normalized to more readily discern differences in MAGE‐A3/6 degradation upon nutrient deprivation.

Data information: Asterisks indicate ***P < 0.001. ns indicates P > 0.05 using two‐way ANOVA.

Figure 4. The CRL4‐DCAF12 E3 ubiquitin ligase degrades MAGE‐A3/6

1. A

   Tandem affinity purification of MAGE‐A3 from HEK293 cells identified DDB1, DCAF5, and DCAF12 as binding partners. Mascot score, number of peptides identified, and percent coverage are shown.

2. B

   MAGE‐A3 interacts with DCAF12, but not DCAF5. HeLa cells were transfected with the indicated constructs (Myc‐MAGE‐A3 and HA‐DCAF5 or HA‐DCAF12). Two days later, cells were treated with complete media or EBSS for 4 h. Cells were treated in the presence of 1 μM MLN4924 to stabilize MAGE‐A3. Cell extracts were subjected to anti‐Myc IP.

3. C

   Nutrient deprivation increases endogenous DCAF12 binding to MAGE‐A3. HeLa cells were transfected with Myc‐vector or Myc‐MAGE‐A3 for 48 h. Cells were then treated with 10 μM MG132 in complete media, EBSS, or EBSS containing 20 μg/ml insulin for 6 h. Cell extracts were subjected to anti‐Myc IP.

4. D, E

   Knockdown of DCAF12, but not DCAF5, altered MAGE‐A3/6 protein levels and partially rescued its instability upon nutrient deprivation. HeLa cells were treated with control, DCAF5, or DCAF12 siRNAs for 72 h before incubation in complete media or EBSS for the indicated times with or without MLN4924 (1 μM).

5. F

   Knockdown of DCAF12 with individual siRNAs blocks MAGE‐A3/6 degradation upon nutrient deprivation. HeLa cells were treated with the indicated siRNAs for 72 h before incubation in EBSS for the indicated times. Note, time 0 samples between siRNA groups were normalized to more readily discern differences in MAGE‐A3/6 degradation upon nutrient deprivation.

6. G

   Ubiquitination of MAGE‐A3/6 upon nutrient deprivation is dependent on DCAF12. A375 parental or DCAF12 KO cells were treated with MG132 (10 μM) in complete media or EBSS for 6 h. Cell extracts were incubated with control agarose or agarose‐TUBE2 to isolate ubiquitinated proteins. Input and pulldown samples were probed for the indicated proteins.

Figure 5. Specific MAGE‐A proteins are regulated by the nutrient‐sensitive CRL4‐DCAF12 ligase

1. Identification of DCAF12 substrates. Control (WT) or DCAF12 knockout (KO) A375 cells were subjected to quantitative TMT proteomics to identify potential DCAF12 targets. MAGE‐A proteins (shown in blue) are stabilized in DCAF12 knockout cells.

2. Knockout of DCAF12 rescues degradation of MAGE‐A proteins in A375 cells. DCAF12 KO A375 cells were treated with complete media or EBSS before quantitative TMT proteomics. Note MAGE‐A genes are not significantly altered by EBSS in DCAF12 KO cells.

3. DCAF12 target proteins are differentially affected by EBSS compared to remainder of the proteome. DCAF12 targets (n = 33) identified in (A) show significantly greater decrease upon EBSS treatment compared with non‐DCAF12‐altered genes (n = 8,243). Average with standard deviation is shown with statistical analysis by Student's t‐test. Asterisks *** indicate P < 0.001 (Student's t‐test).

4. MAGE‐A2, MAGE‐A3, MAGE‐A6, and MAGE‐A12, but not other MAGE‐A genes, contain a DCAF12 degron motif (C‐term –EE*).

5. Overexpression of DCAF12 decreases wild‐type MAGE‐A3, but not MAGE‐A3 degron mutant. The indicated constructs were expressed for 48 h in HeLa cells before immunoblotting. The MAGE‐A3 degron mutant was created as previously described 42, in which DNYNEPKANQ* was added to the extreme C‐term to obscure the DCAF12 degron motif.

6. Analysis of proteomics data (A, B) indicates that MAGE‐A1, MAGE‐A4, MAGE‐A10, and MAGE‐A11 are not regulated by nutrient deprivation or DCAF12. Average (n = 3) with standard deviation is shown with statistical analysis by Student's t‐test. Asterisks *** indicate P < 0.001 (Student's t‐test).

7. MAGE‐A10 and MAGE‐A11 are not altered upon nutrient deprivation or DCAF12 deletion. A375 parental or DCAF12 KO cells were treated with EBSS for the indicated times. Cell lysates were probed for the indicated proteins.

Figure 6. Degradation of MAGE‐A3/6 by DCAF12 is important for autophagy induction upon nutrient deprivation

1. A, B

   U2OS GFP‐LC3 cells were transfected with the indicated siRNAs for 72 h before treatment with complete media or EBSS for 6 h. Cells were then fixed, imaged, and the number of GFP‐LC3 puncta were quantitated per cell. Representative images (A) and quantitation of > 50 cells from three independent experiments (B) are shown as mean ± SD. Scale bar indicates 50 μm.

2. C, D

   A375 parental or DCAF12 KO cells were transfected with the indicated siRNAs for 72 h before treatment with complete media or EBSS for 6 h. Cells were then fixed, endogenous LC3 was immunostained, and the number of LC3 puncta was quantitated per cell. Representative images (C) and quantitation of > 50 cells from three independent experiments (D) are shown as mean ± SD. Scale bar indicates 20 μm.

3. E

   A375 parental or DCAF12 KO cells were treated as described in (C). Cell lysates were collected and immunoblotted for the indicated antibodies to confirm MAGE‐A3/6 depletion and monitor the autophagy adaptor/substrate p62/SQSTM1.

Data information: Asterisks indicate *P < 0.05; ***P < 0.001. ns indicates P > 0.05 using Student's t‐test.