The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.
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.
Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.Show MeSH
Related in: MedlinePlus
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).
Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.