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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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The essential function of human CK1δ is in ribosome maturation. (A) Cell proliferation (as determined by doubling times) of control MDA-MB-231 cells, or of MDA-MB-231 cells where hLtv1 was silenced by Dox-directed induction of hLtv1 shRNA for 3 d, before addition of 30 nM SR3029 or vehicle for another 3 d. (B) Western blot analyses established ∼80% depletion of Ltv1 after 3 d of Ltv1 knockdown. (C) Proliferation of MDA-MB-231 cells engineered to inducibly overexpress wild-type hLtv1, hLtv1-S/D, or hLtv1-S/A after Dox treatment for 3 d. (D) Annexin V/DAPI staining combined with FACS analysis of control MDA-MB-231 cells and those depleted of hLtv1 in the presence or absence of 100 nM SR-3029. All measurements in A, C, and D were done in triplicate, and error bars show the standard deviation of these data.
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fig6: The essential function of human CK1δ is in ribosome maturation. (A) Cell proliferation (as determined by doubling times) of control MDA-MB-231 cells, or of MDA-MB-231 cells where hLtv1 was silenced by Dox-directed induction of hLtv1 shRNA for 3 d, before addition of 30 nM SR3029 or vehicle for another 3 d. (B) Western blot analyses established ∼80% depletion of Ltv1 after 3 d of Ltv1 knockdown. (C) Proliferation of MDA-MB-231 cells engineered to inducibly overexpress wild-type hLtv1, hLtv1-S/D, or hLtv1-S/A after Dox treatment for 3 d. (D) Annexin V/DAPI staining combined with FACS analysis of control MDA-MB-231 cells and those depleted of hLtv1 in the presence or absence of 100 nM SR-3029. All measurements in A, C, and D were done in triplicate, and error bars show the standard deviation of these data.

Mentions: Human CK1δ and CK1ε have ∼65% sequence identity and 85% sequence similarity to Hrr25, and both have been implicated in 40S ribosome maturation (Zemp et al., 2014). To assess the effect of CK1δ/CK1ε inhibition on ribosome assembly and cell growth, we used the small molecule inhibitor SR-3029, a potent and highly specific inhibitor of CK1δ and CK1ε (Bibian et al., 2013). For our experiments, we used MDA-MB-231 triple-negative human breast cancer cells, which express high levels of CK1δ, are sensitive to knockdown of CK1δ (but not to silencing of CK1ε), and are highly sensitive to SR-3029 (Rosenberg et al., 2015). As expected, treatment of these cells with low concentrations of SR-3029 (30 nM) led to marked and rapid reductions in cell growth (Fig. 6 A). This is consistent with previous findings that both CK1δ and CK1ε have essential functions in human cells (Knippschild et al., 2014).


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)

The essential function of human CK1δ is in ribosome maturation. (A) Cell proliferation (as determined by doubling times) of control MDA-MB-231 cells, or of MDA-MB-231 cells where hLtv1 was silenced by Dox-directed induction of hLtv1 shRNA for 3 d, before addition of 30 nM SR3029 or vehicle for another 3 d. (B) Western blot analyses established ∼80% depletion of Ltv1 after 3 d of Ltv1 knockdown. (C) Proliferation of MDA-MB-231 cells engineered to inducibly overexpress wild-type hLtv1, hLtv1-S/D, or hLtv1-S/A after Dox treatment for 3 d. (D) Annexin V/DAPI staining combined with FACS analysis of control MDA-MB-231 cells and those depleted of hLtv1 in the presence or absence of 100 nM SR-3029. All measurements in A, C, and D were done in triplicate, and error bars show the standard deviation of these data.
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Related In: Results  -  Collection

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fig6: The essential function of human CK1δ is in ribosome maturation. (A) Cell proliferation (as determined by doubling times) of control MDA-MB-231 cells, or of MDA-MB-231 cells where hLtv1 was silenced by Dox-directed induction of hLtv1 shRNA for 3 d, before addition of 30 nM SR3029 or vehicle for another 3 d. (B) Western blot analyses established ∼80% depletion of Ltv1 after 3 d of Ltv1 knockdown. (C) Proliferation of MDA-MB-231 cells engineered to inducibly overexpress wild-type hLtv1, hLtv1-S/D, or hLtv1-S/A after Dox treatment for 3 d. (D) Annexin V/DAPI staining combined with FACS analysis of control MDA-MB-231 cells and those depleted of hLtv1 in the presence or absence of 100 nM SR-3029. All measurements in A, C, and D were done in triplicate, and error bars show the standard deviation of these data.
Mentions: Human CK1δ and CK1ε have ∼65% sequence identity and 85% sequence similarity to Hrr25, and both have been implicated in 40S ribosome maturation (Zemp et al., 2014). To assess the effect of CK1δ/CK1ε inhibition on ribosome assembly and cell growth, we used the small molecule inhibitor SR-3029, a potent and highly specific inhibitor of CK1δ and CK1ε (Bibian et al., 2013). For our experiments, we used MDA-MB-231 triple-negative human breast cancer cells, which express high levels of CK1δ, are sensitive to knockdown of CK1δ (but not to silencing of CK1ε), and are highly sensitive to SR-3029 (Rosenberg et al., 2015). As expected, treatment of these cells with low concentrations of SR-3029 (30 nM) led to marked and rapid reductions in cell growth (Fig. 6 A). This is consistent with previous findings that both CK1δ and CK1ε have essential functions in human cells (Knippschild et al., 2014).

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