Phosphorylation of STAT5b by breast tumor kinase
While phosphorylation of Y699 is required for transcriptional activity, three tyrosines located in the transactivation domain can also be phosphorylated and modulate STAT5b activity [23]. These tyrosines are either positive (Y725) or negative (Y740 and Y743) regulators of STAT5b activity, and their phosphorylations are mediated by c-Src (Y699 and Y725) and the EGFR (Y699, Y725, Y740, and Y743) [12,27]. We investigated the ability of Brk to phosphorylate each of the four identified tyrosine sites, starting with the required activating tyrosine, Y699. MEF5-/- cells were transfected with STAT5b along with Brk constructs, and STAT5b immunoprecipitations were analyzed using the antiphospho-Y699 STAT5b-specific antibody.
Both Brk and Y447F Brk were able to mediate the phosphorylation of Y699, while it was not detectable in vector cells or K219M Brk-transfected cells (Figure 2a). To determine whether Brk mediates the phosphorylation of tyrosines other than Y699, MEF5-/- cells were transfected with Brk along with STAT5b mutants in which all but one of the four tyrosines was mutated. Brk induced detectable tyrosine phosphorylation of wildtype STAT5b and the Y725/740/743F mutant (with intact Y699), confirming the results with the antiphospho-Y699-specific antibody. None of the other STAT5b mutant constructs, however, were phosphorylated by Brk (Figure 2b). These results demonstrate that Brk primarily mediates Y699 phosphorylation on STAT5b and not the three transactivation domain tyrosines.
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.

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