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SCF β-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration.

Zhong J, Shaik S, Wan L, Tron AE, Wang Z, Sun L, Inuzuka H, Wei W - Oncotarget (2013)

Bottom Line: Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate.Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration.Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

ABSTRACT
Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.

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CKIδ is involved in the regulation of MTSS1 protein stability mediated by SCFβ-TRCP(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with Myc-MTSS1, Flag-β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. (B) IB analysis of WCL derived from 293T cells transfected with Myc-MTSS1 and/or Myc-CKIδ together with Flag-WT—β-TRCP1 or Flag-R474A—β-TRCP1. (C) IB analysis of WCL and immunoprecipitates (IP) derived from 293T cells transfected with GST-MTSS1 and Myc-tagged versions of the indicated CKI isoforms. (D) IB analysis of HeLa cells that were infected with shRNA specific for GFP or the indicated CKI isoforms, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of HeLa cells treated with the CKI inhibitor D4476 at the indicated concentrations for 12 hours. (F) IB analysis of WCL and IP derived from HeLa cells that were infected with shGFP, shCKIδ-A or shCKIδ-B, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Flag—β-TRCP1 and/or Myc-MTSS1 as indicated. (G) HeLa cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Myc-MTSS1, Flag—β-TRCP1. 20 hours post-transfection, the cells were split into 60-mm dishes before being treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (H) Quantification of the band intensities in G. Myc-MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3).
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Figure 3: CKIδ is involved in the regulation of MTSS1 protein stability mediated by SCFβ-TRCP(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with Myc-MTSS1, Flag-β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. (B) IB analysis of WCL derived from 293T cells transfected with Myc-MTSS1 and/or Myc-CKIδ together with Flag-WT—β-TRCP1 or Flag-R474A—β-TRCP1. (C) IB analysis of WCL and immunoprecipitates (IP) derived from 293T cells transfected with GST-MTSS1 and Myc-tagged versions of the indicated CKI isoforms. (D) IB analysis of HeLa cells that were infected with shRNA specific for GFP or the indicated CKI isoforms, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of HeLa cells treated with the CKI inhibitor D4476 at the indicated concentrations for 12 hours. (F) IB analysis of WCL and IP derived from HeLa cells that were infected with shGFP, shCKIδ-A or shCKIδ-B, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Flag—β-TRCP1 and/or Myc-MTSS1 as indicated. (G) HeLa cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Myc-MTSS1, Flag—β-TRCP1. 20 hours post-transfection, the cells were split into 60-mm dishes before being treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (H) Quantification of the band intensities in G. Myc-MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3).

Mentions: As β-TRCP only recognizes its substrates when they are properly phosphorylated by one or a combination of kinase(s) [32, 33], we sought to identify the upstream kinase that phosphorylates MTSS1 to trigger its destruction by β-TRCP. In this regard, both CKIδ and GSK3β have been previously identified as critical players in phosphorylating the targeted proteins of β-TRCP for ubiquitin-mediated protein degradation [34, 35]. Hence, to determine the specific kinase involved in the degradation of MTSS1, we transfected HeLa and 293T cells with HA-MTSS1 and Flag-β-TRCP1 along with CKIδ or GSK3β and further analyzed MTSS1 levels by western blot analysis. Interestingly, we found that CKIδ, but not GSK3β, efficiently promoted the degradation of MTSS1 (Figure 3A and Supplementary Figure S3A). Furthermore, MG132, a 26S proteasome inhibitor, completely prevented MTSS1 degradation mediated by CKIδ and β-TRCP1, suggesting the involvement of the 26S proteasome in this process (Figure 3A and Supplementary Figure S3A). Importantly, the mutant β-TRCP1 (R474A), which is unable to interact with MTSS1 (Figure 1B), failed to promote MTSS1 degradation in the presence of CKIδ (Figure 3B and Supplementary Figure S3B), indicating a critical role for the C-terminal substrate-binding WD40 repeat motif in β-TRCP1-mediated destruction of MTSS1.


SCF β-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration.

Zhong J, Shaik S, Wan L, Tron AE, Wang Z, Sun L, Inuzuka H, Wei W - Oncotarget (2013)

CKIδ is involved in the regulation of MTSS1 protein stability mediated by SCFβ-TRCP(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with Myc-MTSS1, Flag-β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. (B) IB analysis of WCL derived from 293T cells transfected with Myc-MTSS1 and/or Myc-CKIδ together with Flag-WT—β-TRCP1 or Flag-R474A—β-TRCP1. (C) IB analysis of WCL and immunoprecipitates (IP) derived from 293T cells transfected with GST-MTSS1 and Myc-tagged versions of the indicated CKI isoforms. (D) IB analysis of HeLa cells that were infected with shRNA specific for GFP or the indicated CKI isoforms, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of HeLa cells treated with the CKI inhibitor D4476 at the indicated concentrations for 12 hours. (F) IB analysis of WCL and IP derived from HeLa cells that were infected with shGFP, shCKIδ-A or shCKIδ-B, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Flag—β-TRCP1 and/or Myc-MTSS1 as indicated. (G) HeLa cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Myc-MTSS1, Flag—β-TRCP1. 20 hours post-transfection, the cells were split into 60-mm dishes before being treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (H) Quantification of the band intensities in G. Myc-MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3).
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Figure 3: CKIδ is involved in the regulation of MTSS1 protein stability mediated by SCFβ-TRCP(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HeLa cells transfected with Myc-MTSS1, Flag-β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. (B) IB analysis of WCL derived from 293T cells transfected with Myc-MTSS1 and/or Myc-CKIδ together with Flag-WT—β-TRCP1 or Flag-R474A—β-TRCP1. (C) IB analysis of WCL and immunoprecipitates (IP) derived from 293T cells transfected with GST-MTSS1 and Myc-tagged versions of the indicated CKI isoforms. (D) IB analysis of HeLa cells that were infected with shRNA specific for GFP or the indicated CKI isoforms, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. (E) IB analysis of HeLa cells treated with the CKI inhibitor D4476 at the indicated concentrations for 12 hours. (F) IB analysis of WCL and IP derived from HeLa cells that were infected with shGFP, shCKIδ-A or shCKIδ-B, followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Flag—β-TRCP1 and/or Myc-MTSS1 as indicated. (G) HeLa cells were infected with the indicated shRNA constructs followed by selection with 1 μg/ml puromycin for three days to eliminate the non-infected cells. The various generated HeLa cell lines were then transfected with Myc-MTSS1, Flag—β-TRCP1. 20 hours post-transfection, the cells were split into 60-mm dishes before being treated with 20 μg/ml CHX. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. (H) Quantification of the band intensities in G. Myc-MTSS1 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n= 3).
Mentions: As β-TRCP only recognizes its substrates when they are properly phosphorylated by one or a combination of kinase(s) [32, 33], we sought to identify the upstream kinase that phosphorylates MTSS1 to trigger its destruction by β-TRCP. In this regard, both CKIδ and GSK3β have been previously identified as critical players in phosphorylating the targeted proteins of β-TRCP for ubiquitin-mediated protein degradation [34, 35]. Hence, to determine the specific kinase involved in the degradation of MTSS1, we transfected HeLa and 293T cells with HA-MTSS1 and Flag-β-TRCP1 along with CKIδ or GSK3β and further analyzed MTSS1 levels by western blot analysis. Interestingly, we found that CKIδ, but not GSK3β, efficiently promoted the degradation of MTSS1 (Figure 3A and Supplementary Figure S3A). Furthermore, MG132, a 26S proteasome inhibitor, completely prevented MTSS1 degradation mediated by CKIδ and β-TRCP1, suggesting the involvement of the 26S proteasome in this process (Figure 3A and Supplementary Figure S3A). Importantly, the mutant β-TRCP1 (R474A), which is unable to interact with MTSS1 (Figure 1B), failed to promote MTSS1 degradation in the presence of CKIδ (Figure 3B and Supplementary Figure S3B), indicating a critical role for the C-terminal substrate-binding WD40 repeat motif in β-TRCP1-mediated destruction of MTSS1.

Bottom Line: Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate.Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration.Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

ABSTRACT
Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.

Show MeSH
Related in: MedlinePlus