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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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Constitutively active BRK downregulates Dok1 protein expression.(A) Immunobloting analysis of total cell lysates from HEK-293 stable cell lines is showing the expression of GFP alone' GFP-BRK-WT and GFP-BRK-YF (top panel), BRK (middle panel) and phosphorylated tyrosines (bottom panel). β-tubulin served as a loading control. (B) Immunobloting analysis of endogenous Dok1 in the stable HEK293 sublines. Expression of Dok1 was examined by immunoblotting analysis. (C) Characterization of cell proliferation in response to BRK-WT and BRK-YF. The P-values were determined for control and stably transfected cells and set at ***P≥0.0001, **P≥0.001 and *P≥0.05 for statistical significance.
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pone-0087684-g005: Constitutively active BRK downregulates Dok1 protein expression.(A) Immunobloting analysis of total cell lysates from HEK-293 stable cell lines is showing the expression of GFP alone' GFP-BRK-WT and GFP-BRK-YF (top panel), BRK (middle panel) and phosphorylated tyrosines (bottom panel). β-tubulin served as a loading control. (B) Immunobloting analysis of endogenous Dok1 in the stable HEK293 sublines. Expression of Dok1 was examined by immunoblotting analysis. (C) Characterization of cell proliferation in response to BRK-WT and BRK-YF. The P-values were determined for control and stably transfected cells and set at ***P≥0.0001, **P≥0.001 and *P≥0.05 for statistical significance.

Mentions: The inverse correlation between BRK and Dok1 prompted us to further investigate whether BRK activation and over-expression could modulate the expression of Dok1 protein. In addition, previous studies have shown that oncogenic tyrosine kinases such as p210bcr-abl and v-Src downregulate Dok-1 in a kinase activity-dependent manner [44]. Since we recently reported that constitutively active BRK (BRK-Y447F) promotes tumor formation [28], we examined whether BRK-Y447F, like oncogenic Src, could downregulate endogenous Dok1. We used HEK 293 cells as a model to study the interaction between BRK and Dok1, since HEK 293 cells express high levels of Dok1, but express no endogenous BRK (Figure 4A). We generated three HEK 293 cell lines stably expressing GFP (empty control vector), GFP-BRK WT or GFP-BRK-YF by retroviral transduction. All stable cell lines expressed the transgene as determined by immunoblotting with the anti-GFP antibody (Figure 5A). Immunoblotting with anti-BRK confirmed the expression of GFP-BRK WT and Y447F and also validated the absence of BRK in HEK 293 cells. The BRK-transduced cells displayed elevated levels of phosphorylation of cellular targets, as visualized with an anti-phosphotyrosine antibody, PY20. Furthermore, as expected BRK-Y447F-transduced cells displayed activities that were significantly higher than those of BRK-WT (Figure 5A). We therefore evaluated the expression of Dok1 in all transduced cell lines and the parental control cell line and observed a significant reduction in the levels of Dok1 protein in the cells transduced with constitutively active BRK-Y447F compared to those in the BRK-WT, GFP alone, and in the parental cells (Figure 5A, bottom panels). Since Dok1 is a tumor suppressor and we observed a dramatic difference between the effects of BRK-WT and BRK-Y447F on Dok1 expression, we evaluated the growth rates of the stable cell lines. We found that the BRK-Y447F-transduced cells displayed significantly higher growth rates than the cells transduced with either BRK-WT or GFP alone (Figure 5B). Taken together, our data indicate that the catalytic activation of BRK is critical in its ability to downregulate endogenous Dok1 and that the observed suppression of Dok1 may contribute to BRK-promoted cell growth.


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)

Constitutively active BRK downregulates Dok1 protein expression.(A) Immunobloting analysis of total cell lysates from HEK-293 stable cell lines is showing the expression of GFP alone' GFP-BRK-WT and GFP-BRK-YF (top panel), BRK (middle panel) and phosphorylated tyrosines (bottom panel). β-tubulin served as a loading control. (B) Immunobloting analysis of endogenous Dok1 in the stable HEK293 sublines. Expression of Dok1 was examined by immunoblotting analysis. (C) Characterization of cell proliferation in response to BRK-WT and BRK-YF. The P-values were determined for control and stably transfected cells and set at ***P≥0.0001, **P≥0.001 and *P≥0.05 for statistical significance.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087684-g005: Constitutively active BRK downregulates Dok1 protein expression.(A) Immunobloting analysis of total cell lysates from HEK-293 stable cell lines is showing the expression of GFP alone' GFP-BRK-WT and GFP-BRK-YF (top panel), BRK (middle panel) and phosphorylated tyrosines (bottom panel). β-tubulin served as a loading control. (B) Immunobloting analysis of endogenous Dok1 in the stable HEK293 sublines. Expression of Dok1 was examined by immunoblotting analysis. (C) Characterization of cell proliferation in response to BRK-WT and BRK-YF. The P-values were determined for control and stably transfected cells and set at ***P≥0.0001, **P≥0.001 and *P≥0.05 for statistical significance.
Mentions: The inverse correlation between BRK and Dok1 prompted us to further investigate whether BRK activation and over-expression could modulate the expression of Dok1 protein. In addition, previous studies have shown that oncogenic tyrosine kinases such as p210bcr-abl and v-Src downregulate Dok-1 in a kinase activity-dependent manner [44]. Since we recently reported that constitutively active BRK (BRK-Y447F) promotes tumor formation [28], we examined whether BRK-Y447F, like oncogenic Src, could downregulate endogenous Dok1. We used HEK 293 cells as a model to study the interaction between BRK and Dok1, since HEK 293 cells express high levels of Dok1, but express no endogenous BRK (Figure 4A). We generated three HEK 293 cell lines stably expressing GFP (empty control vector), GFP-BRK WT or GFP-BRK-YF by retroviral transduction. All stable cell lines expressed the transgene as determined by immunoblotting with the anti-GFP antibody (Figure 5A). Immunoblotting with anti-BRK confirmed the expression of GFP-BRK WT and Y447F and also validated the absence of BRK in HEK 293 cells. The BRK-transduced cells displayed elevated levels of phosphorylation of cellular targets, as visualized with an anti-phosphotyrosine antibody, PY20. Furthermore, as expected BRK-Y447F-transduced cells displayed activities that were significantly higher than those of BRK-WT (Figure 5A). We therefore evaluated the expression of Dok1 in all transduced cell lines and the parental control cell line and observed a significant reduction in the levels of Dok1 protein in the cells transduced with constitutively active BRK-Y447F compared to those in the BRK-WT, GFP alone, and in the parental cells (Figure 5A, bottom panels). Since Dok1 is a tumor suppressor and we observed a dramatic difference between the effects of BRK-WT and BRK-Y447F on Dok1 expression, we evaluated the growth rates of the stable cell lines. We found that the BRK-Y447F-transduced cells displayed significantly higher growth rates than the cells transduced with either BRK-WT or GFP alone (Figure 5B). Taken together, our data indicate that the catalytic activation of BRK is critical in its ability to downregulate endogenous Dok1 and that the observed suppression of Dok1 may contribute to BRK-promoted cell growth.

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