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Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines - PubMed

  • ️Sun Jan 01 2006

Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines

Joon No Lee et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH(2)-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.

Sequence alignment between Insig-1 and Insig-2. Identical residues are shaded. S149 in Insig-1 and S106 and E214 in Insig-2 are highlighted in yellow. The ubiquitination sites identified in Insig-1 (K156 and K158) are indicated by asterisks. Underlined are amino acids 131–207 of Insig-1 and the corresponding sequence of Insig-2.

Fig. 2.
Fig. 2.

Stability of mutant Insig-1 and chimeric proteins between Insig-1 and Insig-2. The chimera constructs between Insig-1 and Insig-2 are illustrated, with blue and red bars representing amino acid sequences derived from Insig-1 and Insig-2, respectively. SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 0.5 μg of wild-type pTK-Insig1-Myc; 0.5 μg of pTK-Insig1-Myc (38–277); 2 μg of pTK-Insig1-Myc (84–277); 1.0 μg of chimera A; 0.5 μg of chimeras B, C, and D; and 0.5 μg of pTK-Insig2-Myc, as indicated. Total plasmid concentration was adjusted to 2 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide (CHX) for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-Myc IgG-9E10. Filters were exposed for 1 min.

Fig. 3.
Fig. 3.

Insig-1 is stabilized by mutations at S149 and A162. (A and B) SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 0.5 μg of indicated plasmids. Total plasmid concentration was adjusted to 2 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide (CHX) for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-Myc IgG-9E10. Filters were exposed for 1 min. (C) SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 1.5 μg of pTK-Scap, and 0.5 μg of wild-type or mutant pTK-Insig1-Myc, as indicated. On day 3, cells were switched to medium B containing 1% hydroxypropyl-β-cyclodextrin. After incubation for 1 h at 37°C, cells were washed twice with PBS, switched to medium B in the absence or presence of sterols (10 μg/ml cholesterol plus 1 μg/ml 25-hydroxycholesterol), and incubated for an additional 5 h at 37°C. Cells were then harvested, and cell lysate was immunoprecipitated (IP) with anti-Myc IgG-9E10 to precipitate transfected Insigs. The supernatant (Sup.) and pellet fractions of the immunoprecipitation derived from 0.1 and 0.5 dish of cells, respectively, were subjected to SDS/PAGE and immunoblot analysis with polyclonal anti-Myc and anti-Scap (R139). Filters were exposed for 1 min.

Fig. 4.
Fig. 4.

Stability of mutant Insig-1 containing mutations at S149 and A162. SRD-13A cells were set up, transfected with indicated plasmids, harvested, and analyzed in exactly the same way as that described in the legend for Fig. 3B.

Fig. 5.
Fig. 5.

Stability of mutant Insig-2 and chimeric proteins between Insig-1 and Insig-2. The chimera constructs between Insig-1 and Insig-2 are illustrated, with blue and red bars representing amino acid sequences derived from Insig-1 and Insig-2, respectively. SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 0.3 μg of wild-type or mutant pTK-Insig2-Myc, 0.5 μg of wild-type or mutant pTK-Insig1-Myc, and 0.5 μg of chimera E and F, as indicated. Total plasmid concentration was adjusted to 2 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide (CHX) for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-Myc antibody. Filters were exposed for 1 min.

Fig. 6.
Fig. 6.

E214 is required for the stability of Insig-2. (A–C) SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 1 μg of mutant pTK-Insig2-Myc in which the tetrapeptide sequence was changed from YECK to YACK or AAAA, or 0.5 μg of other plasmids, as indicated. Total plasmid concentration was adjusted to 2 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot with anti-Myc IgG-9E10. Filters were exposed for 1 min. (D) SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. On day 2, cells in each dish were transfected with 1.5 μg of pTK-Scap, 1.0 μg of mutant pTK-Insig2-Myc in which the tetrapeptide sequence was changed from YECK to YACK or AAAA, and 0.5 μg of other mutant pTK-Insig-2-Myc, as indicated. Total plasmid concentration was adjusted to 2.5 μg per dish by using empty vector pcDNA3.1. On day 3, cells were switched to medium B containing 1% hydroxypropyl-β-cyclodextrin. After incubation for 1 h at 37°C, cells were washed twice with PBS, switched to medium B in the absence or presence of sterols (10 μg/ml cholesterol plus 1 μg/ml 25-hydroxycholesterol), and incubated for an additional 5 h at 37°C. Cells were then harvested, and cell lysate was immunoprecipitated (IP) with anti-Myc IgG-9E10 to precipitate transfected Insigs. The supernatant (Sup.) and pellet fractions of the immunoprecipitation derived from 0.1 and 0.5 dish of cells, respectively, were subjected to SDS/PAGE and immunoblot analysis with polyclonal anti-Myc and anti-Scap (R139). Filters were exposed for 1 min.

Fig. 7.
Fig. 7.

Ubiquitination of wild-type and mutant Insig-1 and Insig-2. SRD-13A cells were set up at 3.2 × 105 per 60-mm dish on day 0. (A) On day 2, cells in each dish were transfected with 0.2 μg of pEF1a-HA-ubiqutin, and 0.2 μg of wild-type or mutant pCMV-Insig1-T7, as indicated. Total plasmid concentration was adjusted to 2.0 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 10 μM MG-132 for 30 min. Cells were harvested, and cell lysate was immunoprecipitated (IP) with monoclonal anti-T7 IgG-coupled agarose beads to precipitate transfected Insigs. The pellet fractions of the immunoprecipitation were subjected to SDS/PAGE and immunoblot analysis with anti-T7 and anti-HA antibody. Filters were exposed for 30 sec. (B) On day 2, the cells in each dish were transfected with 0.2 μg of wild-type or mutant pCMV-Insig1-T7, as indicated. Total plasmid concentration was adjusted to 2.0 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide (CHX) for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-T7 antibody. Filters were exposed for 30 sec. (C) On day 2, cells in each dish were transfected with 0.2 μg of pEF1a-HA-ubiqutin, 0.3 μg of wild-type pCMV-Insig-2-T7, 1.0 μg of mutant pCMV-Insig-2-T7 containing E214A mutation, and 0.3 μg of other mutant pCMV-Insig-2-T7, as indicated. Total plasmid concentration was adjusted to 3.0 μg per dish by using empty vector pcDNA3.1. Cells were then treated, harvested, and analyzed as described for A. (D) On day 2, cells in each dish were transfected with the same amount of wild-type or mutant pCMV-Insig2-T7 as shown for C. Total plasmid concentration was adjusted to 3.0 μg per dish by using empty vector pcDNA3.1. On day 3, cells were treated with 50 μM cycloheximide (CHX) for the indicated time. Cells were then harvested, and cell lysate was subjected to SDS/PAGE and immunoblot analysis with anti-T7 antibody. Filters were exposed for 30 sec.

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