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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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Dok1 inhibits BRK-induced cell proliferation and migration.(A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and **P≥0.001 and ***P≥0.0001.
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pone-0087684-g008: Dok1 inhibits BRK-induced cell proliferation and migration.(A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and **P≥0.001 and ***P≥0.0001.

Mentions: Dok1 is a tumor suppressor and several studies have concurred that the overexpression of Dok1 suppresses cell proliferation and migration [45], [48], [59], [60]. Ectopic expression of Dok1 has been shown to inhibit cell proliferation and transformation induced by oncogenic tyrosine kinases, including the p210bcr-abl and Src family kinases [57], [60]. Previously, we along with others showed that BRK overexpression and activation enhanced cell proliferation, cell migration and tumor formation [26], [28], [33], [61], [62], [63]. To test whether Dok1 can also modulate the oncogenic properties of BRK, we evaluated the effect of Dok1 on BRK-induced cell proliferation and migration. HEK 293 cells stably expressing GFP alone, GFP-BRK-WT and GFP-BRKY447F were infected with adenoviruses expressing mCherry-Dok1 (Figure 8A). In the absence of mCherry-Dok1, cells stably expressing GFP-BRKY447F displayed a significantly higher growth rate compared with GFP-BRK-WT expressing cells as well as the control cell lines (Figure 8B). Remarkably, the introduction of Dok1 resulted in a dramatic decrease in the rate of growth of the BRK-Y447F-transduced cells, similar to the levels exhibited by the control and BRK-WT cells (Figure 8C). Similar results were obtained with BRK-negative cells lines MDA-MB-231 stably expressing BRK variants BRK-Y447F or BRK-WT (Figure S4 in File S1). These data indicate that the overexpression of Dok1 suppresses BRK-induced cell proliferation.


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)

Dok1 inhibits BRK-induced cell proliferation and migration.(A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and **P≥0.001 and ***P≥0.0001.
© Copyright Policy
Related In: Results  -  Collection

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pone-0087684-g008: Dok1 inhibits BRK-induced cell proliferation and migration.(A) HEK 293 stable sub-cell lines were transduced with mCherry-Dok1 using adenoviral vector. Cellular proteins were detected in total cell lysates by immunoblotting analysis with anti-BRK, anti-Dok1, and anti-phosphotyrosine antibodies. β-tubulin served as a loading control. (B & C) HEK 293 stable cells were transduced with or without mCherry-Dok1adeno-vector and were monitored for cell proliferation. (D & E) Cell migration determined by the healing of a fixed wound area induced in the different HEK 293 stable transfectant cells. The percentage of open area at 24 h is plotted. (F & G) Cell migration analysis was performed with the indicated stable cell lines expressing mCherry-Dok1 or an empty vector. The assay was based on the rate of wound closure in the scratched cells. The percentage of open area at 24 hours is plotted. The migration assay was performed in three independent experiments. Data are means ± standard errors. Statistics: and **P≥0.001 and ***P≥0.0001.
Mentions: Dok1 is a tumor suppressor and several studies have concurred that the overexpression of Dok1 suppresses cell proliferation and migration [45], [48], [59], [60]. Ectopic expression of Dok1 has been shown to inhibit cell proliferation and transformation induced by oncogenic tyrosine kinases, including the p210bcr-abl and Src family kinases [57], [60]. Previously, we along with others showed that BRK overexpression and activation enhanced cell proliferation, cell migration and tumor formation [26], [28], [33], [61], [62], [63]. To test whether Dok1 can also modulate the oncogenic properties of BRK, we evaluated the effect of Dok1 on BRK-induced cell proliferation and migration. HEK 293 cells stably expressing GFP alone, GFP-BRK-WT and GFP-BRKY447F were infected with adenoviruses expressing mCherry-Dok1 (Figure 8A). In the absence of mCherry-Dok1, cells stably expressing GFP-BRKY447F displayed a significantly higher growth rate compared with GFP-BRK-WT expressing cells as well as the control cell lines (Figure 8B). Remarkably, the introduction of Dok1 resulted in a dramatic decrease in the rate of growth of the BRK-Y447F-transduced cells, similar to the levels exhibited by the control and BRK-WT cells (Figure 8C). Similar results were obtained with BRK-negative cells lines MDA-MB-231 stably expressing BRK variants BRK-Y447F or BRK-WT (Figure S4 in File S1). These data indicate that the overexpression of Dok1 suppresses BRK-induced cell proliferation.

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