Ras Regulates the Association of Serum Response Factor and CCAAT/Enhancer-binding Protein β

Abstract

The serum response element (SRE) is a promoter element essential for transcriptional activation of immediate early genes, such as c-fos and early growth response-1, by mitogenic signals. Several transcription factors bind the SRE, including the serum response factor (SRF), the ternary complex factor, and the CCAAT/ enhancer-binding protein ␤ (C/EBP␤). The C/EBP␤ mRNA encodes three translation products of 38, 35, and 20 kDa. p35-C/EBP␤ activates transcription of the SRE in an SRF-dependent fashion, whereas p20-C/EBP␤, which initiates at an internal in-frame methionine, lacks a transactivation domain and inhibits transcription. We show that SRF and C/EBP␤ interact in vivo through the DNA binding domain of SRF and the C terminus of C/EBP␤ common to p35/38 and p20. Therefore, like the ternary complex factor, C/EBP␤ may be recruited to the SRE not only by binding to the DNA, which is not a high affinity site, but also by proteinprotein interactions with SRF. Strikingly, in both the mammalian two-hybrid assay and in vivo coimmunoprecipitations, the association of SRF and p35-C/EBP␤ but not p20-C/EBP␤ is dramatically stimulated by activated Ras. Furthermore, mutation of the threonine within a mitogen-activated protein kinase consensus motif in the C terminus of C/EBP␤ eliminates the response to Ras. These results suggest a new mechanism by which mitogenic signals may influence transcription activity of the SRE by selectively promoting protein-protein interactions between SRF and the transactivator p35-C/EBP␤.The c-fos proto-oncogene is a prototypic member of the set of "immediate early" genes whose transcription is rapidly activated in the absence of protein synthesis by mitogenic signals (1). Within the promoter of the c-fos gene is a 26-bp 1 regulatory sequence, the serum response element (SRE), that mediates the rapid and transient induction of the c-fos gene by serum and growth factors (2). The SRE binds a ubiquitous transcription factor, the serum response factor (SRF), which is essential but not sufficient for serum and growth factor activation of SRE (3-5). SRF is an obligatory member of a multiprotein complex that occupies the SRE and serves as a convergence point for multiple signal transduction pathways. Thus, growth factors that activate the Ras-Raf-extracellular signal-regulated kinase cascade and phorbol esters target a family of transcription factors containing an N-terminal Ets domain that form a ternary complex with SRF and SRE DNA (6). c-fos promoter mutants that cannot bind the ternary complex factor (TCF) remain responsive to serum induction through a second TCFindependent pathway that still requires SRF and can involve certain Rho family GTPases (5,7,8). The role of accessory factors in the TCF-independent pathway has yet to be completely defined.We have observed that maximal TCF-independent serum induction of the SRE requires SRF and another transcription factor, C/EBP␤ (9, 10). C/EBP␤, which binds to the SRE overlapping and immediately 3Ј to SRF, is a member of the basic leucine zipper family of t...

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals

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C/EBPβ Participates in Regulating Transcription of the p53 Gene in Response to Mitogen Stimulation

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