The Pit-1 Homeodomain and β-Domain Interact with Ets-1 and Modulate Synergistic Activation of the Rat Prolactin Promoter

Abstract

Pit-1/GHF-1 is a pituitary-specific, POU homeodomain transcription factor required for development of somatotroph, lactotroph, and thyrotroph cell lineages and regulation of the temporal and spatial expression of the growth hormone, prolactin (PRL), and thyrotropin-␤ genes. Synergistic interaction of Pit-1 with a member of the Ets family of transcription factors, Ets-1, has been shown to be an important mechanism regulating basal and Ras-induced lactotroph-specific rat (r) PRL promoter activity. Pit-1␤/GHF-2, an alternatively spliced isoform containing a 26-amino acid insert (␤-domain) within its transcription-activation domain, physically interacts with Ets-1 but fails to synergize. By using a series of Pit-1 internal-deletion constructs in a transient transfection protocol to reconstitute rPRL promoter activity in HeLa cells, we have determined that the functional and physical interaction of Pit-1 and Ets-1 is mediated via the POU homeodomain, which is common to both Pit-1 and Pit-1␤. Although the Pit-1 homeodomain is both necessary and sufficient for direct binding to Ets-1 in a DNA-independent manner, an additional interaction surface was mapped to the ␤-domain, specific to the Pit-1␤ isoform. Thus, the unique transcriptional properties of Pit-1 and Pit-1␤ on the rPRL promoter may be due to the formation of functionally distinct complexes of these two Pit-1 isoforms with Ets-1.

Ras Signaling and Transcriptional Synergy at a Flexible Ets-1/Pit-1 Composite DNA Element Is Defined by the Assembly of Selective Activation Domains

“…The plasmids pCGN2-Pit-1, pCGN2-Pit-1⌬2-80, and pCGN2-Pit-1⌬2-45 express amino-terminally hemagglutinin-tagged Pit-1 and amino-terminal TAD deletion mutants under the control of the CMV promoter (13,30) and were generously provided by Dr. David Gordon, University of Colorado Health Sciences Center. The wild-type (WT) pGex Pit-1 (199 -291) expression vector (13) contains the homeodomain of rat Pit-1 fused in-frame to glutathione S-transferase in a modified pGex 2TK vector, pGexDFGK, incorporating the multiple cloning site derived from pCGN2, constructed and provided by Dr. David Gordon, University of Colorado Health Sciences Center. Plasmid pSV-Ras contains the T24 bladder carcinoma Harvey Ras valine 12 mutant oncogene (V-12 Ras) (10).…”

“…On day 2, plasmid DNA for transfection was prepared by mixing 0.8 l/well of Enhancer and 2.5 l/well Effectene reagent diluted in 30 l of EC buffer/well. Where indicated, DNA concentrations of the Pit-1 and Ets constructs were adjusted to give approximately equal levels of expression as determined previously (13). At 24 h following transfection, the cells were harvested and firefly luciferase and Renilla luciferase activity were determined on a Dynex microtiter plate luminometer using the Dual luciferase reporter assay system (Promega).…”

“…6-fold synergy) (3). In addition, Ets-1 and Pit-1 have been shown to bind physically to each other in dilute solution and in the absence of DNA by utilizing the Ets-1 regulatory III (RIII) transcriptional activation domain (TAD) and Pit-1 homeodomain (3,12,13). Finally, Thr-82 of chicken p68 c-Ets-1 was identified as a MAP kinase phosphorylation site, and sitespecific mutation of Thr-82 to alanine resulted in loss of the Ets-1-mediated enhancement of the Ras response (14).…”

“…In addition to its role in DNA binding, the homeodomain of Pit-1 is necessary and sufficient for physical interaction with Ets-1. Although it is unclear what precise role the physical interaction of Ets-1/Pit-1 plays in the synergistic activation of the rPRL promoter, it is intriguing to note that the ␤-domain of the Pit-1␤ isoform also interacts with this Ets-1-specific region (13).…”

Signal transduction and the Ets family of transcription factors

“…The prolactin promoter contains a RRE that is composed of a Pit1 binding site juxtaposed to an Ets-binding site. Ets1 is necessary for maximal synergistic expression of prolactin and this expression is dependent upon Ras-MAPK phosphorylation of Ets1 (Bradford et al, 1995(Bradford et al, , 1996(Bradford et al, , 2000Kimura et al, 2000). Both factors serve to engender speci®city from a general signal transduction pathway, thus allowing for unique responses depending on the cell type.…”