GHF-1/Pit-1 Functions as a Cell-specific Integrator of Ras Signaling by Targeting the Ras Pathway to a Composite Ets-1/GHF-1 Response Element

Andrew P Bradford,Kerry E Conrad,Phat H Tran,Michael C Ostrowski,Arthur Gutierrez-Hartmann

doi:10.1074/jbc.271.40.24639

Abstract

Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/GHF-1 binding site located between positions -217 and -190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes.

Highlights

The p21 Ras proto-oncogene is a critical component of a network of signaling pathways that mediate the control of cell growth, metabolism, and differentiation [1] Signals initiated at transmembrane receptors are transduced via Ras and propagated, by a phosphorylation cascade, to the nucleus, resulting in changes in the activity of specific transcription factors [2, 3]
We have proposed that an Ets binding site (EBS) adjacent to a GHF-1 binding site (footprint IV (FPIV)), spanning positions Ϫ217 to Ϫ190, functions as a composite rat prolactin (rPRL) promoter Raf response element (RRE)
We utilized a series of site-specific mutations and deletions in the proximal rPRL promoter to precisely map the principal and physiologically relevant RRE to the composite Ets-1/GHF-1 binding site spanning positions Ϫ217 to Ϫ190

Summary

Introduction

The p21 Ras proto-oncogene is a critical component of a network of signaling pathways that mediate the control of cell growth, metabolism, and differentiation [1] Signals initiated at transmembrane receptors are transduced via Ras and propagated, by a phosphorylation cascade, to the nucleus, resulting in changes in the activity of specific transcription factors [2, 3]. The resulting pA3(mEBS)rPRLluc and pA3(mFPIV)rPRLluc reporter constructs containing mutations in the Ets and GHF-1 binding sites, respectively, were transiently transfected in the presence of activated Ras or Raf to determine the effect of the mutations on rPRL promoter activation.

Results

Conclusion