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Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

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UBR5 ubiquitinates CDK9 in vitro. Ubiquitination of baculovirus-expressed CDK9 carrying a His tag was obtained in the presence of the enzymes E1, E2 (UbcH5b), and E3 (affinity purified UBR5). The reactions were incubated with ubiquitin (Ub), ubiquitin aldehyde, and AMP-PNP as described under “Experimental Procedures.” Higher molecular weight ubiquitinated forms of CDK9-His were detected by immunoblotting with anti-His or anti-CDK9 antibodies (left panel). To confirm that the higher molecular weight bands contained ubiquitinated forms of CDK9, replicate reactions were analyzed by immunoblotting with an anti-ubiquitin antibody (right panel). WB, Western blotting.
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Figure 2: UBR5 ubiquitinates CDK9 in vitro. Ubiquitination of baculovirus-expressed CDK9 carrying a His tag was obtained in the presence of the enzymes E1, E2 (UbcH5b), and E3 (affinity purified UBR5). The reactions were incubated with ubiquitin (Ub), ubiquitin aldehyde, and AMP-PNP as described under “Experimental Procedures.” Higher molecular weight ubiquitinated forms of CDK9-His were detected by immunoblotting with anti-His or anti-CDK9 antibodies (left panel). To confirm that the higher molecular weight bands contained ubiquitinated forms of CDK9, replicate reactions were analyzed by immunoblotting with an anti-ubiquitin antibody (right panel). WB, Western blotting.

Mentions: Because a previous report indicated that endogenous CDK9 molecules are ubiquitinated (30), it was legitimate to speculate that its interaction partner, UBR5, may be involved in this modification. To test this hypothesis, we first performed in vitro ubiquitination assays using TAP affinity purified UBR5 and CDK9 purified from insect cells as a substrate. When the complete set of components was used in the ubiquitination reaction, a robust polyubiquitination of CDK9 was observed, with the appearance of slower migrating high molecular weight species in lanes that contain the complete reaction mixture (Fig. 2). These forms contained ubiquitinated CDK9 molecules, because they were detected with both antibodies directed toward CDK9 (anti-tag and anti-CDK9), as well as with the anti-ubiquitin antibody. When one of the components was omitted (E1, E2, CDK9, or UBR5), no ubiquitination was observed. These results show that UBR5 directly catalyzes the ubiquitination of CDK9 in vitro.


Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

UBR5 ubiquitinates CDK9 in vitro. Ubiquitination of baculovirus-expressed CDK9 carrying a His tag was obtained in the presence of the enzymes E1, E2 (UbcH5b), and E3 (affinity purified UBR5). The reactions were incubated with ubiquitin (Ub), ubiquitin aldehyde, and AMP-PNP as described under “Experimental Procedures.” Higher molecular weight ubiquitinated forms of CDK9-His were detected by immunoblotting with anti-His or anti-CDK9 antibodies (left panel). To confirm that the higher molecular weight bands contained ubiquitinated forms of CDK9, replicate reactions were analyzed by immunoblotting with an anti-ubiquitin antibody (right panel). WB, Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037613&req=5

Figure 2: UBR5 ubiquitinates CDK9 in vitro. Ubiquitination of baculovirus-expressed CDK9 carrying a His tag was obtained in the presence of the enzymes E1, E2 (UbcH5b), and E3 (affinity purified UBR5). The reactions were incubated with ubiquitin (Ub), ubiquitin aldehyde, and AMP-PNP as described under “Experimental Procedures.” Higher molecular weight ubiquitinated forms of CDK9-His were detected by immunoblotting with anti-His or anti-CDK9 antibodies (left panel). To confirm that the higher molecular weight bands contained ubiquitinated forms of CDK9, replicate reactions were analyzed by immunoblotting with an anti-ubiquitin antibody (right panel). WB, Western blotting.
Mentions: Because a previous report indicated that endogenous CDK9 molecules are ubiquitinated (30), it was legitimate to speculate that its interaction partner, UBR5, may be involved in this modification. To test this hypothesis, we first performed in vitro ubiquitination assays using TAP affinity purified UBR5 and CDK9 purified from insect cells as a substrate. When the complete set of components was used in the ubiquitination reaction, a robust polyubiquitination of CDK9 was observed, with the appearance of slower migrating high molecular weight species in lanes that contain the complete reaction mixture (Fig. 2). These forms contained ubiquitinated CDK9 molecules, because they were detected with both antibodies directed toward CDK9 (anti-tag and anti-CDK9), as well as with the anti-ubiquitin antibody. When one of the components was omitted (E1, E2, CDK9, or UBR5), no ubiquitination was observed. These results show that UBR5 directly catalyzes the ubiquitination of CDK9 in vitro.

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

Show MeSH