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Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

Bottom Line: Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion.Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly.These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

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Hrr25-dependent release of Ltv1 from pre-40S ribosomes. (A) Co-sedimentation assays. Western blot analysis of AFs in the bound (pellet [P]) and released (supernatant [S]) fractions of the purified pre-40S ribosome from cells containing wild-type (WT-Hrr25) or 1NA-PP1-sensitive mutant (Hrr25-I82G) of Hrr25. (B) Native gel assay for Ltv1 release. Western blot analyses of native gels are shown. The position of ribosome-bound and free Ltv1 is identified by Rio2TAP-purified ribosomes and recombinant Ltv1.
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fig3: Hrr25-dependent release of Ltv1 from pre-40S ribosomes. (A) Co-sedimentation assays. Western blot analysis of AFs in the bound (pellet [P]) and released (supernatant [S]) fractions of the purified pre-40S ribosome from cells containing wild-type (WT-Hrr25) or 1NA-PP1-sensitive mutant (Hrr25-I82G) of Hrr25. (B) Native gel assay for Ltv1 release. Western blot analyses of native gels are shown. The position of ribosome-bound and free Ltv1 is identified by Rio2TAP-purified ribosomes and recombinant Ltv1.

Mentions: The kinase Hrr25 plays roles in pre-40S maturation, and Hrr25 depletion leads to reduced levels of phosphorylated Enp1, Ltv1, and Rps3 (Schäfer et al., 2006; Zemp et al., 2014). To test if Hrr25 kinase activity was required for release of Ltv1 or Enp1 from pre-40S subunits, we developed a release assay. Purified pre-40S ribosomes were incubated with nanomolar concentrations of ATP, and release of Ltv1 and Enp1 was monitored by sedimentation through a sucrose cushion as described above (Fig. 3 A) or by native gel shift analysis (Fig. 3 B). Both methods show that addition of ATP to pre-40S ribosomes leads to dissociation of Ltv1 and Enp1 (Fig. 3, A and B). The Rio2 ATPase is also dissociated, which is expected as its ATPase activity is linked to its dissociation from pre-40S ribosomes (Ferreira-Cerca et al., 2012). In contrast, other AFs remain bound.


Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

Hrr25-dependent release of Ltv1 from pre-40S ribosomes. (A) Co-sedimentation assays. Western blot analysis of AFs in the bound (pellet [P]) and released (supernatant [S]) fractions of the purified pre-40S ribosome from cells containing wild-type (WT-Hrr25) or 1NA-PP1-sensitive mutant (Hrr25-I82G) of Hrr25. (B) Native gel assay for Ltv1 release. Western blot analyses of native gels are shown. The position of ribosome-bound and free Ltv1 is identified by Rio2TAP-purified ribosomes and recombinant Ltv1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362465&req=5

fig3: Hrr25-dependent release of Ltv1 from pre-40S ribosomes. (A) Co-sedimentation assays. Western blot analysis of AFs in the bound (pellet [P]) and released (supernatant [S]) fractions of the purified pre-40S ribosome from cells containing wild-type (WT-Hrr25) or 1NA-PP1-sensitive mutant (Hrr25-I82G) of Hrr25. (B) Native gel assay for Ltv1 release. Western blot analyses of native gels are shown. The position of ribosome-bound and free Ltv1 is identified by Rio2TAP-purified ribosomes and recombinant Ltv1.
Mentions: The kinase Hrr25 plays roles in pre-40S maturation, and Hrr25 depletion leads to reduced levels of phosphorylated Enp1, Ltv1, and Rps3 (Schäfer et al., 2006; Zemp et al., 2014). To test if Hrr25 kinase activity was required for release of Ltv1 or Enp1 from pre-40S subunits, we developed a release assay. Purified pre-40S ribosomes were incubated with nanomolar concentrations of ATP, and release of Ltv1 and Enp1 was monitored by sedimentation through a sucrose cushion as described above (Fig. 3 A) or by native gel shift analysis (Fig. 3 B). Both methods show that addition of ATP to pre-40S ribosomes leads to dissociation of Ltv1 and Enp1 (Fig. 3, A and B). The Rio2 ATPase is also dissociated, which is expected as its ATPase activity is linked to its dissociation from pre-40S ribosomes (Ferreira-Cerca et al., 2012). In contrast, other AFs remain bound.

Bottom Line: Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion.Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly.These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

Show MeSH
Related in: MedlinePlus