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Differential Utilization of Transcription Activation Subdomains by Distinct Coactivators Regulates Pit-1 Basal and Ras Responsiveness

Departments of Medicine (M.D.J., A.G.-H.), Biochemistry and Molecular Genetics (A.G.-H.), and Clinical Pharmacy (D.L.D., A.J.), University of Colorado Health Sciences Center, Aurora, Colorado 80045; and Department of Physiology (S.E.D., P.M.), University of Kentucky College of Medicine, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Arthur Gutierrez-Hartmann, University of Colorado Health Sciences Center, P.O. Box 6511, Mail Stop 8106, Aurora, Colorado 80045. E-mail: a.gutierrez-hartmann{at}uchsc.edu and dawn.duval{at}uchsc.edu.

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1ß, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1–45), and a basal and Ras-responsive region, R2 (AA46–80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1ßTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50–70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60–80. In contrast, GAL4-Pit-1ßTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the ß-domain, we inserted the ß-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1ß, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, ß-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61–71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1ß TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a ß-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.