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RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells.

Hsin JP, Li W, Hoque M, Tian B, Manley JL - Elife (2014)

Bottom Line: Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y).Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro.Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, United States.

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Related in: MedlinePlus

Western blotting analysis and quantification.(A) GST-CTD proteins were phosphorylated by Abl tyrosine kinase for indicated time in vitro. Reactions were analyzed by western blotting with indicated antibodies. (B) Y1F cells grown in the absence of tet were treated with 50 nM epoxomicin or 5 µM MG132 for 4 hr. Cell lysates were analyzed by western blotting as in Figure 3D. The levels of full-length Rpb1 were quantified using ImageJ, and ratios of full-length Rpb1 to degraded Rpb1 (IIB) and actin were presented. N = 3. Error bars display standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.008
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fig3s1: Western blotting analysis and quantification.(A) GST-CTD proteins were phosphorylated by Abl tyrosine kinase for indicated time in vitro. Reactions were analyzed by western blotting with indicated antibodies. (B) Y1F cells grown in the absence of tet were treated with 50 nM epoxomicin or 5 µM MG132 for 4 hr. Cell lysates were analyzed by western blotting as in Figure 3D. The levels of full-length Rpb1 were quantified using ImageJ, and ratios of full-length Rpb1 to degraded Rpb1 (IIB) and actin were presented. N = 3. Error bars display standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.008

Mentions: We next asked whether phosphorylation of GST-CTD affects its stability in the proteasome assay. For this, we first used a recombinant c-Abl derivative to phosphorylate GST-CTD. This resulted in conversion of a fraction of the GST-CTD to a low-mobility, Tyr1-P isoform, although the majority remained unphosphorylated (Figure 3C, lane 1, Figure 3—figure supplement 1A), consistent with the processive phosphorylation by c-Abl observed previously (Duyster et al., 1995). We then performed the proteasome assay described above using c-Abl-phosphorylated GST-CTD (Figure 3C). Strikingly, the Tyr1 hyperphosphorylated GST-CTD (top panel, upper band, and lower panel) was resistant to degradation (lane 2), while the remaining unphosphorylated GST-CTD (top panel, bottom band) was degraded. Addition of 0.01% SDS again promoted degradation of unphosphorylated GST-CTD, but the Tyr1-P isoform remained resistant (lane 3). Significantly, GST-CTD phosphorylated by the Ser5/Ser7 kinase CDK7, which converted essentially all of the substrate to the hyperphosphorylated form, was not protected from degradation (lanes 4–6), indicating a specific role of Tyr1-P in preventing proteasomal degradation.


RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells.

Hsin JP, Li W, Hoque M, Tian B, Manley JL - Elife (2014)

Western blotting analysis and quantification.(A) GST-CTD proteins were phosphorylated by Abl tyrosine kinase for indicated time in vitro. Reactions were analyzed by western blotting with indicated antibodies. (B) Y1F cells grown in the absence of tet were treated with 50 nM epoxomicin or 5 µM MG132 for 4 hr. Cell lysates were analyzed by western blotting as in Figure 3D. The levels of full-length Rpb1 were quantified using ImageJ, and ratios of full-length Rpb1 to degraded Rpb1 (IIB) and actin were presented. N = 3. Error bars display standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4042873&req=5

fig3s1: Western blotting analysis and quantification.(A) GST-CTD proteins were phosphorylated by Abl tyrosine kinase for indicated time in vitro. Reactions were analyzed by western blotting with indicated antibodies. (B) Y1F cells grown in the absence of tet were treated with 50 nM epoxomicin or 5 µM MG132 for 4 hr. Cell lysates were analyzed by western blotting as in Figure 3D. The levels of full-length Rpb1 were quantified using ImageJ, and ratios of full-length Rpb1 to degraded Rpb1 (IIB) and actin were presented. N = 3. Error bars display standard deviation.DOI:http://dx.doi.org/10.7554/eLife.02112.008
Mentions: We next asked whether phosphorylation of GST-CTD affects its stability in the proteasome assay. For this, we first used a recombinant c-Abl derivative to phosphorylate GST-CTD. This resulted in conversion of a fraction of the GST-CTD to a low-mobility, Tyr1-P isoform, although the majority remained unphosphorylated (Figure 3C, lane 1, Figure 3—figure supplement 1A), consistent with the processive phosphorylation by c-Abl observed previously (Duyster et al., 1995). We then performed the proteasome assay described above using c-Abl-phosphorylated GST-CTD (Figure 3C). Strikingly, the Tyr1 hyperphosphorylated GST-CTD (top panel, upper band, and lower panel) was resistant to degradation (lane 2), while the remaining unphosphorylated GST-CTD (top panel, bottom band) was degraded. Addition of 0.01% SDS again promoted degradation of unphosphorylated GST-CTD, but the Tyr1-P isoform remained resistant (lane 3). Significantly, GST-CTD phosphorylated by the Ser5/Ser7 kinase CDK7, which converted essentially all of the substrate to the hyperphosphorylated form, was not protected from degradation (lanes 4–6), indicating a specific role of Tyr1-P in preventing proteasomal degradation.

Bottom Line: Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y).Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro.Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, United States.

Show MeSH
Related in: MedlinePlus