Signal transducer and activator of transcription 5b: a new target of breast tumor kinase/protein tyrosine kinase 6

Abstract

Introduction: Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b.

Methods: To determine whether Brk can phosphorylate STAT5b, transient transfection and in vitro kinase assays were performed. Luciferase reporter assays were used to measure Brk-induced STAT5b transcriptional activity. siRNA technology was utilized to investigate the biological significance of Brk-induced activation of STAT5b in breast cancer cell models.

Results: Phosphospecific antibodies, mutational analysis, and in vitro kinase assays demonstrated that Brk specifically mediated STAT5b phosphorylation at the activating tyrosine, Y699. Transient transfection of Brk into the Brk-negative BT-549 breast cancer cell line enhanced STAT5b transcriptional activity, as measured by a STAT5-specific luciferase reporter. Furthermore, overexpression of kinase active c-Src enhanced Brk-induced STAT5b transcriptional activity. In Brk-positive breast cancer cell lines BT-20 and SKBr3, knockdown of Brk protein or of STAT5b protein using siRNA methodology resulted in a decrease in DNA synthesis. Knockdown of Brk and STAT5b together did not further decrease DNA synthesis compared with each alone, suggesting that Brk and STAT5b converge on the same pathway, ultimately leading to cellular proliferation.

Conclusion: Our studies demonstrate that Brk phosphorylates STAT5b on Y699, leading to increased STAT5b transcriptional activity. Furthermore, analysis of DNA synthesis suggests that STAT5b and Brk are converging upon the same proproliferative signaling pathway in breast cancer cells. We propose that Brk, like other tyrosine kinases, signals downstream to STAT5b to mediate proliferation of breast cancer cells. These results further establish STAT5b as well as Brk as potential targets for breast cancer therapy.

Figure 1

Breast tumor kinase mediates tyrosine phosphorylation of signal transducers and activators of transcription. Breast tumor kinase (Brk) mediates tyrosine phosphorylation of signal transducer and activator of transcription (STAT)3, STAT5a, and STAT5b. STAT5a/b knockout mouse embryo fibroblast (MEF5^-/-) cells were transfected with (a) STAT3, (b) STAT5a, or (c) STAT5b along with either pRc (vector), Brk, K219M Brk (kinase inactive), or Y477F Brk (constitutively active). Immunoprecipitations (IPs) for STAT3, STAT5a, or STAT5b were performed using antibodies specifically directed against each STAT and were analyzed by immunoblotting with antibodies directed toward STAT3, STAT5a, STAT5b (middle), or phosphotyrosine (top). Total lysates were analyzed for the expression of the Brk construct by immunoblotting with anti-Brk antibody (bottom).

Figure 2

Signal transducer and activator of transcription 5b tyrosine phosphorylation and transcriptional activation. (a) Mouse embryo fibroblast (MEF5^-/-) cells were transfected with signal transducer and activator of transcription 5b (STAT5b) along with pRc (vector), breast tumor kinase (Brk), K219M, or Y447F. Immunoprecipitations (IPs) were performed using antibodies specifically against STAT5b and were analyzed by immunoblotting with antibodies directed toward phospho-Y699 STAT5b (top) or STAT5b (bottom). (b) MEF5^-/- cells were transfected with various STAT5b constructs along with constitutively active Y447F Brk. Immunoprecipitations (IPs) were performed as above and were analyzed via immunoblotting with antiphosphotyrosine antibody (top) or anti-STAT5b antibody (bottom). (c) MEF5^-/- were transfected with the STAT5-specific Spi2.1 promoter luciferase construct and either His-vector, His-STAT5b, or His-Y699F with or without Brk. Luciferase activity was measured and normalized to total protein. Values from four independent experiments performed in triplicate reported as the fold induction over His, pRc ± standard error: His, pRc (1.00 ± 0.00) and Brk (1.05 ± 0.39); STAT5b, pRc (28.89 ± 5.51) and Brk (75.48 ± 25.79); Y699F, pRc (1.17 ± 0.14) and Brk (0.93 ± 0.27). Student's t test was used to determine statistical significance between STAT5b pRc and STAT5b Brk. *P = 0.0123.

Figure 3

In vitro Y699 phosphorylation by breast tumor kinase. In vitro Y699 phosphorylation of signal transducer and activator of transcription 5b (STAT5b) by breast tumor kinase (Brk). Purified recombinant GST, GST-STAT5b, or GST-Y699F were incubated with (+) or without (-) purified recombinant Brk in reaction buffer containing 20 nM ATP at 30°C for 30 minutes. An equal volume of 2 × Laemmli buffer was added to end the reaction and the samples were run on SDS-PAGE. Gels were analyzed via (a) silver stain and (b) by immunoblotting with an antiphospho-Y699 STAT5b-specific antibody.

Figure 4

Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.

Figure 5

Breast tumor kinase mediates endogenous signal transducer and activator of transcription 5b transcriptional activity. (a) BT-549 cells were transfected with the Spi2.1 promoter luciferase construct along with pRc, breast tumor kinase (Brk), K219M, or Y447F expression vectors. Luciferase activity was measured and normalized to total protein. Values from four independent experiments carried out in triplicate were reported as average luciferase/protein ± standard error: pRc (1,859.37 ± 592.24); Brk (10,031.94 ± 1,359.68); K219M (3,109.82 ± 777.57), Y477F (12,878.57 ± 2,464.26). Student's t test was used to determine statistical significance between pRc and Brk, and between pRc and K219M. *P < 0.0001, • P = 0.0430. (b) BT-549 cells were transfected with the Spi2.1 promoter luciferase construct along with pRc, Brk, or K219M, and either pcDNA, c-Src, or K-Src expression vectors. Luciferase activity was measured and normalized to total protein. Values from four independent experiments carried out in triplicate were reported as the average fold induction over pcDNA, pRc ± SE: pcDNA, pRc (1.00 ± 0.00), Brk (2.92 ± 0.39), and K219M (1.36 ± 0.13); c-Src, pRc (1.10 ± 0.12), Brk (5.01 ± 0.38), and K219M (1.24 ± 0.18); K-Src, pRc (0.99 ± 0.19) and Brk (2.76 ± 0.89). Student's t test was used to determine statistical significance between pcDNA pRc and pcDNA Brk, between pcDNA pRc and pcDNA K219M, and between pRc Brk and c-Src Brk. *P = 0.0027, • P = 0.0322, ◆ P < 0.0086.

Figure 6

Breast tumor kinase and signal transducer and activator of transcription 5b roles in DNA synthesis. Knockdown of breast tumor kinase (Brk) and/or signal transducer and activator of transcription 5b (STAT5b) in SKBr3 cells (a, b) or BT-20 cells (c, d) was performed using the Dharmacon siGenome SMARTpool duplex for each protein as per the manufacturer's instructions. (a, c) Seventy-two hours after transfection of the siRNA, cells were lysed. Total lysates were analyzed via immunoblotting with anti-STAT5b (top), anti-Brk (middle), or anti-β-actin (bottom) antibodies. (b, d) Sixty-six hours after transfection of the siRNA, bromodeoxyuridine (BrdU) was added to the medium for 6 hours. The cells were fixed and stained with a fluorescent antibody against BrdU. Cells were scored for BrdU incorporation and graphed. Between 160 and 200 cells were counted for each treatment group. (b) Average percentage of BrdU incorporation ± standard error from three independent experiments performed in triplicate for the SKBr3 cells: media (21.54 ± 0.16), siLuc (24.98 ± 2.78), siBrk (12.37 ± 1.65), siSTAT5b (12.37 ± 0.51), siBrk/siSTAT5b (12.39 ± 0.21). Student's t test was utilized to determine statistical significance between media and siBrk, siSTAT5b, or siBrk/siSTAT5b, and between siLuc and siBrk, siSTAT5b, or siBrk/siSTAT5b. *P ≤ 0.0050, • P < 0.0025. (d) Average percentage of BrdU incorporation ± standard error from three independent experiments performed in triplicate for the BT-20 cells: media (25.28 ± 0.55), siLuc (26.87 ± 0.52), siBrk (18.97 ± 0.32), siSTAT5b (18.87 ± 0.03), siBrk/siSTAT5b (17.37 ± 0.88). Student's t test was utilized to determine statistical significance between media and siBrk, siSTAT5b, or siBrk/siSTAT5b, and between siLuc and siBrk, siSTAT5b, or siBrk/siSTAT5b. *P ≤ 0.0016, • P < 0.0007.