PMC:2246177 / 23448-27080 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2246177","sourcedb":"PMC","sourceid":"2246177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2246177","text":"Breast tumor kinase mediates STAT5b activity in breast cancer cells\nTo examine the ability of Brk to mediate the transcriptional activity of endogenous STAT5b in a breast cancer cell line, the BT-549 cells, which have no detectable endogenous Brk, were utilized. The STAT5-specific Spi2.1 luciferase reporter with or without Brk was transfected into the BT-549 cells. While there was a detectable basal level of transcriptional activity, Brk significantly increased endogenous STAT5b transcriptional activity, as did the constitutively active Brk (Y447F) (Figure 5a). Although not nearly as effective, the kinase inactive Brk (K219M) did significantly increase the STAT5b luciferase activity compared with vector alone. This result is not necessarily unexpected since a role of the kinase inactive Brk in other cell models has been reported (see Discussion).\nFigure 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 \u003c 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 \u003c 0.0086. Since Brk and c-Src are both able to phosphorylate STAT5b on Y699, the potential cooperation between Brk and c-Src was investigated. As previously, Brk dramatically enhanced STAT5b transcriptional activity, and the K219M Brk was also able to significantly enhance this activity (Figure 5b). Unexpectedly, exogenous overexpression of c-Src did not enhance STAT5b transcriptional activity (Figure 5b). c-Src overexpression enhanced Brk-induced STAT5b transcriptional activity, however, indicating that Brk and c-Src together can enhance STAT5b transcriptional activity. The kinase activity of Brk was necessary for this collaboration as the addition of c-Src to K219M Brk did not enhance STAT5b activity. Likewise, the kinase inactive c-Src (K-Src) failed to significantly enhance Brk-induced STAT5b transcriptional activity, suggesting that the kinase activity of c-Src is also necessary for this cooperation between Brk and c-Src. While Brk and c-Src individually have different effects on STAT5b transcriptional activity, these two tyrosine kinases together increase STAT5b transcriptional activity. These data suggest that Brk and c-Src do not merely substitute for one another in activating STAT5b, but rather that Brk can function independently as well as together with c-Src.","divisions":[{"label":"Title","span":{"begin":0,"end":67}},{"label":"Figure caption","span":{"begin":859,"end":2351}}],"tracks":[]}