Abstract
In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a beta-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.
Highlights
In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy
There have been numerous studies following the kinetics of signal transducers and activators of transcription (STAT) tyrosine phosphorylation and DNA binding as a function of cytokine activation, there are relatively few reports in which the subcellular distribution of STATs following activation by either cytokines or nonreceptor tyrosine kinases such as src has been investigated
The tyrosine phosphorylation and DNA binding activity was initially compared with the nuclear localization of STAT5A and STAT5B following both Prl and src activation
Summary
Prolactin; PrlR, Prl receptor; STAT, signal transducers and activators of transcription; IL, interleukin; CAT, chloramphenicol acetyltransferase; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4Ј,6-diamidino-2-phenylindole; RIPA, radio immunoprecipitation assay. The targeted knockout of the individual genes in mice has suggested that they play essential but often redundant roles in the physiological responses associated with Prl [25] Despite their homology, there is some evidence suggesting that STAT5A and STAT5B may be differentially activated [26] and even exhibit distinct DNA binding specificities [27]. Src kinase activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B, but unlike prolactin induction, nuclear translocation of only STAT5B but not STAT5A was observed This selective nuclear translocation appears to be mediated via the carboxylterminal sequences in STAT5B and was not prevented but instead stimulated by a dominant-negative kinase-inactive mutant of JAK2. Family that results in the selective nuclear translocation of STAT5B and possibly activation of unique gene targets
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