Limits...
The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.

Shalev M, Aviram R, Adamovich Y, Kraut-Cohen J, Shamia T, Ben-Dor S, Golik M, Asher G - Nucleic Acids Res. (2014)

Bottom Line: Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts.Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP).Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Show MeSH

Related in: MedlinePlus

BMAL1/CLOCK interact with REV-ERBα. (A) Schematic representation of the evolutionary conservation of the PXDLS motif (green) in REV-ERBα. Mice were sacrificed at Zeitgerber 4 (ZT4), 4 h after lights were turned on in the animal facility, livers were harvested and nuclear extracts were prepared. Immunoprecipitation (IP) experiments were performed with anti BMAL1 (B) or anti REV-ERBα (C) antibodies. Rap1 antibody was used as negative control. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (D) BMAL1-Flag and REV-ERBα were expressed in NIH3T3 cells; protein lysates were prepared and immunoprecipitated with Flag or Rap1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (E) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα. Recombinant His-BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. Molecular Weight (MW). *Non-specific band. An arrow indicates the full length BMAL1. **A degradation product of recombinant BMAL1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231743&req=5

Figure 5: BMAL1/CLOCK interact with REV-ERBα. (A) Schematic representation of the evolutionary conservation of the PXDLS motif (green) in REV-ERBα. Mice were sacrificed at Zeitgerber 4 (ZT4), 4 h after lights were turned on in the animal facility, livers were harvested and nuclear extracts were prepared. Immunoprecipitation (IP) experiments were performed with anti BMAL1 (B) or anti REV-ERBα (C) antibodies. Rap1 antibody was used as negative control. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (D) BMAL1-Flag and REV-ERBα were expressed in NIH3T3 cells; protein lysates were prepared and immunoprecipitated with Flag or Rap1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (E) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα. Recombinant His-BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. Molecular Weight (MW). *Non-specific band. An arrow indicates the full length BMAL1. **A degradation product of recombinant BMAL1.

Mentions: To uncover the molecular mechanisms underlying the involvement of the PXDLS motif in circadian oscillations, we searched for proteins containing the PXDLS motif among proteins that play a role in circadian rhythmicity. Notably, we found that the motif is evolutionary conserved in the core clock protein, REV-ERBα (Figure 5A), that participates in the repression of Bmal1 promoter (6–9). The conserved PXDLS domain in REV-ERBα on the one hand, and our findings that BMAL1/CLOCK can bind the PXDLS motif on the other hand, prompted us to examine whether REV-ERBα interacts with BMAL1/CLOCK. Thus, we performed co-immunoprecipitation experiments from liver nuclear extracts. Immunoprecipitation of BMAL1 resulted in co-immunoprecipitation of endogenous REV-ERBα and CLOCK (Figure 5B, Supplementary Figures S8A and S9A). Likewise, both endogenous BMAL1 and CLOCK co-immunoprecipitated with REV-ERBα (Figure 5C, Supplementary Figures S8B and S9B). Similar interaction was detected in NIH3T3 cells, upon co-expression of BMAL1-Flag and REV-ERBα and immunoprecipitation of BMAL1-Flag (Figure 5D). Finally, to test whether this interaction can be recapitulated in vitro, we prepared recombinant BMAL1 and examined its binding to in vitro translated [35S] labeled REV-ERBα. REV-ERBα was specifically pulled down in the presence of recombinant BMAL1 (Figure 5E).


The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.

Shalev M, Aviram R, Adamovich Y, Kraut-Cohen J, Shamia T, Ben-Dor S, Golik M, Asher G - Nucleic Acids Res. (2014)

BMAL1/CLOCK interact with REV-ERBα. (A) Schematic representation of the evolutionary conservation of the PXDLS motif (green) in REV-ERBα. Mice were sacrificed at Zeitgerber 4 (ZT4), 4 h after lights were turned on in the animal facility, livers were harvested and nuclear extracts were prepared. Immunoprecipitation (IP) experiments were performed with anti BMAL1 (B) or anti REV-ERBα (C) antibodies. Rap1 antibody was used as negative control. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (D) BMAL1-Flag and REV-ERBα were expressed in NIH3T3 cells; protein lysates were prepared and immunoprecipitated with Flag or Rap1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (E) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα. Recombinant His-BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. Molecular Weight (MW). *Non-specific band. An arrow indicates the full length BMAL1. **A degradation product of recombinant BMAL1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231743&req=5

Figure 5: BMAL1/CLOCK interact with REV-ERBα. (A) Schematic representation of the evolutionary conservation of the PXDLS motif (green) in REV-ERBα. Mice were sacrificed at Zeitgerber 4 (ZT4), 4 h after lights were turned on in the animal facility, livers were harvested and nuclear extracts were prepared. Immunoprecipitation (IP) experiments were performed with anti BMAL1 (B) or anti REV-ERBα (C) antibodies. Rap1 antibody was used as negative control. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (D) BMAL1-Flag and REV-ERBα were expressed in NIH3T3 cells; protein lysates were prepared and immunoprecipitated with Flag or Rap1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (E) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα. Recombinant His-BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. Molecular Weight (MW). *Non-specific band. An arrow indicates the full length BMAL1. **A degradation product of recombinant BMAL1.
Mentions: To uncover the molecular mechanisms underlying the involvement of the PXDLS motif in circadian oscillations, we searched for proteins containing the PXDLS motif among proteins that play a role in circadian rhythmicity. Notably, we found that the motif is evolutionary conserved in the core clock protein, REV-ERBα (Figure 5A), that participates in the repression of Bmal1 promoter (6–9). The conserved PXDLS domain in REV-ERBα on the one hand, and our findings that BMAL1/CLOCK can bind the PXDLS motif on the other hand, prompted us to examine whether REV-ERBα interacts with BMAL1/CLOCK. Thus, we performed co-immunoprecipitation experiments from liver nuclear extracts. Immunoprecipitation of BMAL1 resulted in co-immunoprecipitation of endogenous REV-ERBα and CLOCK (Figure 5B, Supplementary Figures S8A and S9A). Likewise, both endogenous BMAL1 and CLOCK co-immunoprecipitated with REV-ERBα (Figure 5C, Supplementary Figures S8B and S9B). Similar interaction was detected in NIH3T3 cells, upon co-expression of BMAL1-Flag and REV-ERBα and immunoprecipitation of BMAL1-Flag (Figure 5D). Finally, to test whether this interaction can be recapitulated in vitro, we prepared recombinant BMAL1 and examined its binding to in vitro translated [35S] labeled REV-ERBα. REV-ERBα was specifically pulled down in the presence of recombinant BMAL1 (Figure 5E).

Bottom Line: Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts.Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP).Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Show MeSH
Related in: MedlinePlus