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The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

Ren H, Koo J, Guan B, Yue P, Deng X, Chen M, Khuri FR, Sun SY - Mol. Cancer (2013)

Bottom Line: API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1.Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1.However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiration, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, P R China. chenmingwei@mail.xjtu.edu.cn.

ABSTRACT

Background: The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.

Results: API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

Conclusions: API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

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Both the SCF E3 ligases, β-TrCP and FBXW7, are involved in mediating API-1-induced Mcl-1 degradation. A, H1299 cells were transfected with control (Ctrl) or FBXW7 siRNA and 48 h later were exposed to DMSO or 5 μM API-1 for an additional 4 h. Moreover, WT and FBXW7-KO HCT116 cell lines were treated with 5 μM for 4 h. B, H1299 cells were pre-treated with the indicated concentrations of MLN4924 and then co-treated with the given concentrations of API-1 for an additional 4 h. C and D, H1299 cells were transfected with the given siRNAs for 48 h and then exposed to 5 μM API-1 for another 4 h. After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting. Protein levels were quantified with NIH Image J software and were normalized to actin. E, H1299 cells were transfected with the given siRNAs and after 48 h were harvested for extraction of total RNA. RT-PCR was then conducted to detect the expression of the indicated mRNAs.
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Figure 7: Both the SCF E3 ligases, β-TrCP and FBXW7, are involved in mediating API-1-induced Mcl-1 degradation. A, H1299 cells were transfected with control (Ctrl) or FBXW7 siRNA and 48 h later were exposed to DMSO or 5 μM API-1 for an additional 4 h. Moreover, WT and FBXW7-KO HCT116 cell lines were treated with 5 μM for 4 h. B, H1299 cells were pre-treated with the indicated concentrations of MLN4924 and then co-treated with the given concentrations of API-1 for an additional 4 h. C and D, H1299 cells were transfected with the given siRNAs for 48 h and then exposed to 5 μM API-1 for another 4 h. After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting. Protein levels were quantified with NIH Image J software and were normalized to actin. E, H1299 cells were transfected with the given siRNAs and after 48 h were harvested for extraction of total RNA. RT-PCR was then conducted to detect the expression of the indicated mRNAs.

Mentions: Finally, we investigated which E3 ubiquitin ligase is responsible for the GSK3-dependent proteasomal degradation of Mcl-1 induced by API-1. Given the recent studies on the critical role of FBXW7 in mediating GSK3-dependent degradation of Mcl-1 [10,11], we first determined whether this E3 ligase is involved in API-1-induced Mcl-1 degradation. Using the validated FBXW7 siRNA in our previous study [13], we detected slightly less reduction of Mcl-1 in FBXW7 siRNA-transfected cells than in control siRNA-transfected cells (Figure 7A). We further compared the effects of API-1 on Mcl-1 reduction between WT and FBXW7-KO HCT116 cells and generated similar results (Figure 7A) as we observed in the above knockdown experiment. These data collectively suggest that FBXW7 is only partly involved in mediating GSK3-dependent degradation of Mcl-1 by API-1, and that additional E3 ligases(s) are also involved in this process.


The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

Ren H, Koo J, Guan B, Yue P, Deng X, Chen M, Khuri FR, Sun SY - Mol. Cancer (2013)

Both the SCF E3 ligases, β-TrCP and FBXW7, are involved in mediating API-1-induced Mcl-1 degradation. A, H1299 cells were transfected with control (Ctrl) or FBXW7 siRNA and 48 h later were exposed to DMSO or 5 μM API-1 for an additional 4 h. Moreover, WT and FBXW7-KO HCT116 cell lines were treated with 5 μM for 4 h. B, H1299 cells were pre-treated with the indicated concentrations of MLN4924 and then co-treated with the given concentrations of API-1 for an additional 4 h. C and D, H1299 cells were transfected with the given siRNAs for 48 h and then exposed to 5 μM API-1 for another 4 h. After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting. Protein levels were quantified with NIH Image J software and were normalized to actin. E, H1299 cells were transfected with the given siRNAs and after 48 h were harvested for extraction of total RNA. RT-PCR was then conducted to detect the expression of the indicated mRNAs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924345&req=5

Figure 7: Both the SCF E3 ligases, β-TrCP and FBXW7, are involved in mediating API-1-induced Mcl-1 degradation. A, H1299 cells were transfected with control (Ctrl) or FBXW7 siRNA and 48 h later were exposed to DMSO or 5 μM API-1 for an additional 4 h. Moreover, WT and FBXW7-KO HCT116 cell lines were treated with 5 μM for 4 h. B, H1299 cells were pre-treated with the indicated concentrations of MLN4924 and then co-treated with the given concentrations of API-1 for an additional 4 h. C and D, H1299 cells were transfected with the given siRNAs for 48 h and then exposed to 5 μM API-1 for another 4 h. After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blotting. Protein levels were quantified with NIH Image J software and were normalized to actin. E, H1299 cells were transfected with the given siRNAs and after 48 h were harvested for extraction of total RNA. RT-PCR was then conducted to detect the expression of the indicated mRNAs.
Mentions: Finally, we investigated which E3 ubiquitin ligase is responsible for the GSK3-dependent proteasomal degradation of Mcl-1 induced by API-1. Given the recent studies on the critical role of FBXW7 in mediating GSK3-dependent degradation of Mcl-1 [10,11], we first determined whether this E3 ligase is involved in API-1-induced Mcl-1 degradation. Using the validated FBXW7 siRNA in our previous study [13], we detected slightly less reduction of Mcl-1 in FBXW7 siRNA-transfected cells than in control siRNA-transfected cells (Figure 7A). We further compared the effects of API-1 on Mcl-1 reduction between WT and FBXW7-KO HCT116 cells and generated similar results (Figure 7A) as we observed in the above knockdown experiment. These data collectively suggest that FBXW7 is only partly involved in mediating GSK3-dependent degradation of Mcl-1 by API-1, and that additional E3 ligases(s) are also involved in this process.

Bottom Line: API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1.Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1.However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiration, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, P R China. chenmingwei@mail.xjtu.edu.cn.

ABSTRACT

Background: The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.

Results: API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.

Conclusions: API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Show MeSH
Related in: MedlinePlus