Abstract
Most transcription factors possess at least one long intrinsically disordered transactivation domain that binds to a variety of coactivators and corepressors and plays a key role in modulating the transcriptional activity. Despite the crucial importance of these domains, the structural and functional basis of transactivation remains poorly understood. Here, we focused on activating transcription factor 4 (ATF4)/cAMP response element-binding protein-2, an essential transcription factor for cellular stress adaptation. Bioinformatic sequence analysis of the ATF4 transactivation domain sequence revealed that the first 125 amino acids have noticeably less propensity for structural disorder than the rest of the domain. Using solution nuclear magnetic resonance spectroscopy complemented by a range of biophysical methods, we found that the isolated transactivation domain is predominantly yet not fully disordered in solution. We also observed that a short motif at the N-terminus of the transactivation domain has a high helical propensity. Importantly, we found that the N-terminal region of the transactivation domain is involved in transient long-range interactions with the basic-leucine zipper domain involved in DNA binding. Finally, in vitro phosphorylation assays with the casein kinase 2 show that the presence of the basic-leucine zipper domain is required for phosphorylation of the transactivation domain. This study uncovers the intricate coupling existing between the transactivation and basic-leucine zipper domains of ATF4, highlighting its potential regulatory significance.
Highlights
The activating transcription factor 4 (ATF4), called cAMP response element binding protein-2 (CREB-2), is a transcription factor that mediates cellular gene expression changes in response to different types of cellular stress.[1,2,3] While constitutively expressed at low concentrations, ATF4 can be rapidly induced under particular cell-stress conditions such as endoplasmic reticulum stress[4], amino acid deprivation[5], and oxidative stress.[6]
The primary phosphorylation sites of protein kinase A (PKA)[17], ribosomal S6 kinase 2 (RSK2)[18], casein kinase 2 (CK2)[19], and RET tyrosine kinase[20], among others, are all known to be localized within the transactivation domain (TAD) of ATF4
The basic-leucine zipper inhibitor (bZip) domain which is essential for DNA binding and dimerization of ATF4 is highly conserved among the compared species (Fig. 1B)
Summary
The activating transcription factor 4 (ATF4), called cAMP response element binding protein-2 (CREB-2), is a transcription factor that mediates cellular gene expression changes in response to different types of cellular stress.[1,2,3] While constitutively expressed at low concentrations, ATF4 can be rapidly induced under particular cell-stress conditions such as endoplasmic reticulum stress[4], amino acid deprivation[5], and oxidative stress.[6]. Human ATF4 is a 351 amino acid long protein organized into two functional domains, an N-terminal transactivation domain (TAD, residues 1275) and a C-terminal basic-leucine zipper domain (bZip, residues 276-351). The bZip domain forms an extended -helix encompassing both the basic and leucine-zipper regions in a continuous helical structure.[13] Like many other transactivation domains in transcription factors, the TAD is predicted to be structurally disordered but plays a key role in modulating the activation and degradation of ATF4.14 The N-terminal TAD contains a known binding site for several important binding partners, including p300/CBP associated factor (PCAF)[1] and TrCP.[14,15,16] The TAD is the target of multiple post-translational modifications. The primary phosphorylation sites of protein kinase A (PKA)[17], ribosomal S6 kinase 2 (RSK2)[18], casein kinase 2 (CK2)[19], and RET tyrosine kinase[20], among others, are all known to be localized within the TAD of ATF4
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