Dynamic Trafficking of STAT5 Depends on an Unconventional Nuclear Localization Signal

Abstract

SummarySignal transducer and activator of transcription 5 (STAT5) is crucial for physiological processes that include hematopoiesis, liver metabolism and mammary gland development. However, aberrant continual activity of STAT5 has been causally linked to human leukemias and solid tumor formation. As a regulated transcription factor, precise cellular localization of STAT5 is essential. Conventional nuclear localization signals consist of short stretches of basic amino acids. In this study, we provide evidence that STAT5 nuclear import is dependent on an unconventional nuclear localization signal that functions within the conformation of an extensive coiled-coil domain. Both in vitro binding and in vivo functional assays reveal that STAT5 nuclear import is mediated by the importin-a3/ b1 system independently of STAT5 activation by tyrosine phosphorylation. The integrity of the coiled-coil domain is essential for STAT5 transcriptional induction of the b-casein gene following prolactin stimulation as well as its ability to synergize with the glucocorticoid receptor. The glucocorticoid receptor accumulates in the nucleus in response to prolactin and this nuclear import is dependent on STAT5 nuclear import. STAT5 continually shuttles in and out of the nucleus and live cell imaging demonstrates that STAT5 nuclear export is mediated by both chromosome region maintenance 1 (Crm1)-dependent and Crm1-independent pathways. A Crm1-dependent nuclear export signal was identified within the STAT5 N-terminus. These findings provide insight into the fundamental mechanisms that regulate STAT5 nuclear trafficking and cooperation with the glucocorticoid receptor and provide a basis for clinical intervention of STAT5 function in disease.

STATs get their move on

“…Live cell imaging with cytoplasmic FLIP was used to follow the kinetics of STAT5A-GFP nuclear export in the presence or absence of the Crm1 inhibitor LMB. 97 LMB was found to reduce the kinetics of STAT5A nuclear export, but it did not eliminate its nuclear export. Analysis of fragments of STAT5A capable of mediating nuclear export identified a Crm1-dependent NES in STAT5A N-terminus, and a Crm1-independent NES within the DBD of STAT5A.…”

“…97 The coiled-coil domain of STAT5 is composed of four α-helices. 93 Evaluation of each of these helices tagged with GFP either alone or in combination demonstrated that all four α-helices were required for effective nuclear import.…”

“…93 Evaluation of each of these helices tagged with GFP either alone or in combination demonstrated that all four α-helices were required for effective nuclear import. 97 Although mutation of specific residues in the coiled-coil domain could abrogate nuclear import, STAT5A import is dependent on an unconventional NLS that functions within the conformation of an extensive coiled-coil domain. This domain was used in protein-protein interaction studies, and similar to STAT3, STAT5A binds to the importin-α3 adaptor and STAT5A nuclear import is inhibited with siRNA to importin-α3.…”

“…The specific residues that contribute to the NLS in each STAT appear to be distinct. 79,97,98 For this reason it may be possible to develop specific discriminating inhibitors that can block nuclear import of these individual STATs.…”

Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

“…In support of this idea, ectopic STAT3 activity in vitro can promote AT2 differentiation in the absence of exogenous Dx. Interestingly, GR and STAT proteins have been reported to act together in multiple settings via several molecular mechanisms including joint transcriptional activation and/or repression (Engblom et al, 2007; Langlais et al, 2012) and control of nuclear localisation (Shin and Reich, 2013). …”