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BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Miah S, Goel RK, Dai C, Kalra N, Beaton-Brown E, Bagu ET, Bonham K, Lukong KE - PLoS ONE (2014)

Bottom Line: Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362.We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism.Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

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Activated BRK downregulates Dok1 by reducing its stability.(A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.
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pone-0087684-g007: Activated BRK downregulates Dok1 by reducing its stability.(A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.

Mentions: Our results in Figure 6 led us to the hypothesis that BRK downregulates Dok1 by reducing the stability of Dok1 protein. To investigate this possibility, we examined the relative half-life of endogenous Dok1 protein in the presence or absence of BRK-Y447F. To this end, we treated various BRK-Y447F-transduced HEK 293 cells and control cells at various time points between 1 and 24 hours with cycloheximide (CHX, an inhibitor of protein synthesis). The cells were harvested periodically as indicated in Figure 7A. Western blotting of BRK-Y447F cell lysates showed that Dok1 protein levels were reduced by more than 50%, 75% and 95% at 8, 12 and 24 hours, respectively following treatment with CHX (Figure 7A, left panel). However, in the control samples (Figure 7A, right panel) the initial Dok1 protein level was reduced to half after 12 hours of treatment with CHX and a residual level of about 25% after 24 hours (Figure 7A, right panel). Statistical quantification of the levels of expression of Dok1 is provided in Figure 7A, lower panels. Our data thus indicate that Dok1 exhibited a relatively shorter half-life in cells transduced with BRK-Y447F compared with the control cells. These data indicate that Dok1 is a relatively stable protein with a physiological half-life of about 12 hours and that the stability of the Dok1 protein is compromised in the presence of constitutively active BRK.


BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Miah S, Goel RK, Dai C, Kalra N, Beaton-Brown E, Bagu ET, Bonham K, Lukong KE - PLoS ONE (2014)

Activated BRK downregulates Dok1 by reducing its stability.(A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921129&req=5

pone-0087684-g007: Activated BRK downregulates Dok1 by reducing its stability.(A) HEK 293 cells or HEK 293-BRK-YF stable cell line were treated with a protein synthesis inhibitor cyclohexamide (CHX: 200 µg/ml) for the indicated time points and then the cells were lysed and analyzed by immunoblotting for Dok1, BRK and β-tubulin as a loading control. (B) HEK 293 cells were stably transduced with HEK293-BRK-YF and treated with either a proteosome inhibitor MG132 (10 µM) or the vehicle DMSO as the control, at different time points (above the plot). Cellular proteins were determined in total cell lysates by immunoblotting analysis with anti-Dok1, anti-BRK, anti-phosphotyrosine antibodies. β-tubulin was used as a loading control. (C) Empty vector or V-Src was transiently transfected into HEK293 cells and the cells treated with a proteosome inhibitor MG132 (10 µM) and vehicle control DMSO for the indicated time points. Immunoblotting analysis of total cell lysates was performed to detect Dok1, v-Src, phosphotyrosines and β-tubulin served as a loading control. (D & E) HEK 293 cells were transfected with empty control vector or BRK-YF or v-Src and treated with MG132 (10 µM) and Lactacystin (5 µM) or control vehicle for 8 hours. Then the cell lysates were subjected to immunoblot analysis with anti-Dok1 antibody. β-tubulin as a loading control. (F) HEK293-BRK-YF stable cells were transiently cotransfected with Dok1 and HA-Ubiquitin plasmids and after 12 hours the cells were treated MG132 (10 µM) for an additional 8 hours. The total cell lysates were subjected to immunoprecipitation with anti-Dok1 followed by immunoblotting analysis with anti-HA and anti-Dok1 antibodies. The inputs were analysed as indicated.
Mentions: Our results in Figure 6 led us to the hypothesis that BRK downregulates Dok1 by reducing the stability of Dok1 protein. To investigate this possibility, we examined the relative half-life of endogenous Dok1 protein in the presence or absence of BRK-Y447F. To this end, we treated various BRK-Y447F-transduced HEK 293 cells and control cells at various time points between 1 and 24 hours with cycloheximide (CHX, an inhibitor of protein synthesis). The cells were harvested periodically as indicated in Figure 7A. Western blotting of BRK-Y447F cell lysates showed that Dok1 protein levels were reduced by more than 50%, 75% and 95% at 8, 12 and 24 hours, respectively following treatment with CHX (Figure 7A, left panel). However, in the control samples (Figure 7A, right panel) the initial Dok1 protein level was reduced to half after 12 hours of treatment with CHX and a residual level of about 25% after 24 hours (Figure 7A, right panel). Statistical quantification of the levels of expression of Dok1 is provided in Figure 7A, lower panels. Our data thus indicate that Dok1 exhibited a relatively shorter half-life in cells transduced with BRK-Y447F compared with the control cells. These data indicate that Dok1 is a relatively stable protein with a physiological half-life of about 12 hours and that the stability of the Dok1 protein is compromised in the presence of constitutively active BRK.

Bottom Line: Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362.We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism.Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

ABSTRACT
Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

Show MeSH
Related in: MedlinePlus