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

The PXDLS motif plays a role in BMAL1/CLOCK–REV-ERBα interaction. (A) NIH3T3 cells were transfected with BMAL1-Flag either with wild-type REV-ERBα or with PXDLS mutant REV-ERBα (REV-ERBα P72A) or non-transfected; protein lysates were prepared and immunoprecipitated with Flag antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (B) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα in the presence or absence of recombinant GST-PXDLS*3 or GST-Mut PXDLS*3 peptide. Recombinant BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. (C) NIH3T3 cells were transiently transfected with Bmal1-luciferase reporter either with control empty vector (black) or with wild-type REV-ERBα (red) or PXDLS mutant REV-ERBα (green). Cells were synchronized with a short dexamethasone (Dex) treatment, and real-time bioluminescence recording was performed using the LumiCycle. Amplitudes were quantified using the LumiCycle software. Data are presented on a bar graph as fold change (mean +/− SD, n = 4), P-values <0.001 is marked with **. (D) The expression levels of wild-type REV-ERBα and PXDLS mutant REV-ERBα were determined by SDS-PAGE and IB. U2AF was used as loading control. Molecular Weight (MW). An arrow indicates the full length BMAL1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: The PXDLS motif plays a role in BMAL1/CLOCK–REV-ERBα interaction. (A) NIH3T3 cells were transfected with BMAL1-Flag either with wild-type REV-ERBα or with PXDLS mutant REV-ERBα (REV-ERBα P72A) or non-transfected; protein lysates were prepared and immunoprecipitated with Flag antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (B) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα in the presence or absence of recombinant GST-PXDLS*3 or GST-Mut PXDLS*3 peptide. Recombinant BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. (C) NIH3T3 cells were transiently transfected with Bmal1-luciferase reporter either with control empty vector (black) or with wild-type REV-ERBα (red) or PXDLS mutant REV-ERBα (green). Cells were synchronized with a short dexamethasone (Dex) treatment, and real-time bioluminescence recording was performed using the LumiCycle. Amplitudes were quantified using the LumiCycle software. Data are presented on a bar graph as fold change (mean +/− SD, n = 4), P-values <0.001 is marked with **. (D) The expression levels of wild-type REV-ERBα and PXDLS mutant REV-ERBα were determined by SDS-PAGE and IB. U2AF was used as loading control. Molecular Weight (MW). An arrow indicates the full length BMAL1.

Mentions: To examine the role of the PXDLS motif in the BMAL1/CLOCK–REV-ERBα interactions, we mutated the PXDLS motif of REV-ERBα (REV-ERBα P72A) and examined its binding to BMAL1. Co-immunoprecpitation experiments showed that mutation in the PXDLS motif of REV-ERBα decreased the binding of REV-ERBα to BMAL1 (Figure 6A). To further test the role of the PXDLS motif of REV-ERBα in the interaction, we examined the effect of recombinant PXDLS peptide on the binding of in vitro translated [35S] labeled REV-ERBα, to recombinant BMAL1. The binding of in vitro translated [35S] labeled REV-ERBα to recombinant BMAL1 declined in the presence of excess of PXDLS but not the mutant PXDLS peptide (Figure 6B).


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)

The PXDLS motif plays a role in BMAL1/CLOCK–REV-ERBα interaction. (A) NIH3T3 cells were transfected with BMAL1-Flag either with wild-type REV-ERBα or with PXDLS mutant REV-ERBα (REV-ERBα P72A) or non-transfected; protein lysates were prepared and immunoprecipitated with Flag antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (B) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα in the presence or absence of recombinant GST-PXDLS*3 or GST-Mut PXDLS*3 peptide. Recombinant BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. (C) NIH3T3 cells were transiently transfected with Bmal1-luciferase reporter either with control empty vector (black) or with wild-type REV-ERBα (red) or PXDLS mutant REV-ERBα (green). Cells were synchronized with a short dexamethasone (Dex) treatment, and real-time bioluminescence recording was performed using the LumiCycle. Amplitudes were quantified using the LumiCycle software. Data are presented on a bar graph as fold change (mean +/− SD, n = 4), P-values <0.001 is marked with **. (D) The expression levels of wild-type REV-ERBα and PXDLS mutant REV-ERBα were determined by SDS-PAGE and IB. U2AF was used as loading control. Molecular Weight (MW). An arrow indicates the full length BMAL1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: The PXDLS motif plays a role in BMAL1/CLOCK–REV-ERBα interaction. (A) NIH3T3 cells were transfected with BMAL1-Flag either with wild-type REV-ERBα or with PXDLS mutant REV-ERBα (REV-ERBα P72A) or non-transfected; protein lysates were prepared and immunoprecipitated with Flag antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and IB. (B) Pull down assay with recombinant His-BMAL1 coupled to beads and in vitro translated [35S] labeled REV-ERBα in the presence or absence of recombinant GST-PXDLS*3 or GST-Mut PXDLS*3 peptide. Recombinant BMAL1 was detected by Coomassie stained SDS-PAGE and [35S] labeled REV-ERBα by autoradiography. (C) NIH3T3 cells were transiently transfected with Bmal1-luciferase reporter either with control empty vector (black) or with wild-type REV-ERBα (red) or PXDLS mutant REV-ERBα (green). Cells were synchronized with a short dexamethasone (Dex) treatment, and real-time bioluminescence recording was performed using the LumiCycle. Amplitudes were quantified using the LumiCycle software. Data are presented on a bar graph as fold change (mean +/− SD, n = 4), P-values <0.001 is marked with **. (D) The expression levels of wild-type REV-ERBα and PXDLS mutant REV-ERBα were determined by SDS-PAGE and IB. U2AF was used as loading control. Molecular Weight (MW). An arrow indicates the full length BMAL1.
Mentions: To examine the role of the PXDLS motif in the BMAL1/CLOCK–REV-ERBα interactions, we mutated the PXDLS motif of REV-ERBα (REV-ERBα P72A) and examined its binding to BMAL1. Co-immunoprecpitation experiments showed that mutation in the PXDLS motif of REV-ERBα decreased the binding of REV-ERBα to BMAL1 (Figure 6A). To further test the role of the PXDLS motif of REV-ERBα in the interaction, we examined the effect of recombinant PXDLS peptide on the binding of in vitro translated [35S] labeled REV-ERBα, to recombinant BMAL1. The binding of in vitro translated [35S] labeled REV-ERBα to recombinant BMAL1 declined in the presence of excess of PXDLS but not the mutant PXDLS peptide (Figure 6B).

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