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The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Gotoh T, Vila-Caballer M, Santos CS, Liu J, Yang J, Finkielstein CV - Mol. Biol. Cell (2014)

Bottom Line: We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2.Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes.Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

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The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+myc-hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α-myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc-hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc-Mdm2 (ratio 1:2:5: FLAG-hp53:myc-Mdm2:myc-hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub)n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub)n) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α-myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.
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Figure 2: The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+myc-hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α-myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc-hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc-Mdm2 (ratio 1:2:5: FLAG-hp53:myc-Mdm2:myc-hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub)n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub)n) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α-myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.

Mentions: The Mdm2 protein acts as a bona fide E3-ubiquitin ligase for p53 by binding to its N-terminus and promoting either p53 monoubiquitination and nuclear export or p53 polyubiquitination and degradation through the proteasomal pathway (Honda et al., 1997; Li et al., 2003). Our results show that hPer2 primarily interacts within the C-terminal half of hp53. Thus it is possible that Mdm2 would also be sequestered into the complex. Extracts from HEK293 and HCT116 cells were immunoprecipitated with α-p53 and α-Per2 antibodies, respectively, and complex molecules were detected by immunoblotting (Figure 2A). Consistent with this hypothesis, coimmunoprecipitation and immunoblotting showed that, in both cases and under physiological conditions, Mdm2 associates with hp53/hPer2. This reveals the presence of the hp53/hPer2/Mdm2 endogenous complex in cells (Figure 2A). This result is in line with observations that all three proteins colocalize in similar cellular compartments and thus their interaction could readily occur (Supplemental Figure S2A; Giannakakou et al., 2000; Lohrum et al., 2000; Miyazaki et al., 2001).


The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells.

Gotoh T, Vila-Caballer M, Santos CS, Liu J, Yang J, Finkielstein CV - Mol. Biol. Cell (2014)

The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+myc-hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α-myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc-hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc-Mdm2 (ratio 1:2:5: FLAG-hp53:myc-Mdm2:myc-hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub)n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub)n) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α-myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.
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Figure 2: The hPer2 protein forms a ternary complex with hp53 and Mdm2, controlling the extent of hp53 ubiquitination. (A) HEK293 and HCT116 protein extracts (1 and 2 mg, respectively) were incubated with either α-p53 or α-Per2, respectively, and protein A beads. Rabbit IgG was used as a negative control. Immunoprecipitated complexes were analyzed for the presence of hPer2, hp53, and Mdm2 using specific antibodies (top for HEK293 and bottom for HCT116). Asterisk indicates a nonspecific signal. (B) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hp53, pCS2+FLAG-hp53, pCS2+FLAG-hPer2, pCS2+myc-hPer2, or a combination of plasmids and complexes immunoprecipitated using α-FLAG–coupled beads. Complex components were identified by immunoblotting using α-FLAG and α-myc antibodies (right top and bottom). Input amounts were monitored in cell lysates (20 μg) and shown in the left top and bottom. Results similar to those presented were observed in two independent experiments. (C) In vitro–synthesized myc-hPer2 and FLAG-hp53 proteins were preincubated before the addition of myc-Mdm2 (ratio 1:2:5: FLAG-hp53:myc-Mdm2:myc-hPer2). Samples were then subjected to in vitro ubiquitination, followed by immunoprecipitation of hp53-bound proteins using α-FLAG antibody and protein A beads. Bound proteins were detected by immunoblotting and are indicated with arrows (top and middle). FLAG-hp53(Ub)n forms of hp53 were detected using α-ubiquitin antibody (bottom). Asterisk indicates IgG heavy chain. (D) HEK293 cells were transfected with pCS2+myc-Mdm2, pCS2+myc-hPer2, pCS2+FLAG-hp53, or a combination of plasmids and collected 12 h after treatment with 10 μM MG132. Cell lysates (100 μg) were incubated with α-FLAG and protein A beads and hp53-ubiquitinated complexes (FLAG-hp53(Ub)n) detected using α-ubiquitin antibody (top right). Bound proteins were visualized by immunoblotting using α-myc and α-FLAG antibodies (middle and lower right). An in vitro ubiquitination reaction was performed as described in C and is shown as control and for comparison purposes with the “in cells” result (left). Brackets denote ubiquitinated hp53. A, C, and D show immunoblot data from a single experiment that was repeated three times with similar results.
Mentions: The Mdm2 protein acts as a bona fide E3-ubiquitin ligase for p53 by binding to its N-terminus and promoting either p53 monoubiquitination and nuclear export or p53 polyubiquitination and degradation through the proteasomal pathway (Honda et al., 1997; Li et al., 2003). Our results show that hPer2 primarily interacts within the C-terminal half of hp53. Thus it is possible that Mdm2 would also be sequestered into the complex. Extracts from HEK293 and HCT116 cells were immunoprecipitated with α-p53 and α-Per2 antibodies, respectively, and complex molecules were detected by immunoblotting (Figure 2A). Consistent with this hypothesis, coimmunoprecipitation and immunoblotting showed that, in both cases and under physiological conditions, Mdm2 associates with hp53/hPer2. This reveals the presence of the hp53/hPer2/Mdm2 endogenous complex in cells (Figure 2A). This result is in line with observations that all three proteins colocalize in similar cellular compartments and thus their interaction could readily occur (Supplemental Figure S2A; Giannakakou et al., 2000; Lohrum et al., 2000; Miyazaki et al., 2001).

Bottom Line: We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53's negative regulator, Mdm2.Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes.Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.

View Article: PubMed Central - PubMed

Affiliation: Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

Show MeSH
Related in: MedlinePlus