Identification of Multiple Nuclear Factors That Interact with Cyclic Adenosine 3′,5′-Monophosphate Response Element-Binding Protein and Activating Transcription Factor-2 by Protein-Protein Interactions

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Abstract
Understanding the nature and importance of protein-protein interactions in the mechanisms of eukaryotic gene expression is essential to understanding the normal and aberrant regulation of gene transcription. Using 125I-labeled cAMP response element-binding protein (CREB) and activating transcription factor-2 (ATF-2) recombinant peptides to probe Western blots of HeLa nuclear extracts, we have identified multiple separate nuclear factors that form specific protein-protein interactions with these leucine zipper-containing transcriptional regulatory proteins. The interaction is specific because preincubation of blots with cold homologous protein blocks the binding of labeled protein, whereas preincubation of blots with cold heterologous protein has no effect on labeled protein interactions. Although these studies focus on two specific transactivators, CREB and ATF-2, the approach is of general use for the study of other leucine zipper-containing mammalian transcription factors. Furthermore, in addition to allowing the detection of protein-protein interactions of CREB and ATF-2 with nuclear factors, we have used this strategy to isolate cDNA clones expressing these nuclear proteins. We demonstrate that CREB will form heterodimers with ATF-1, but not ATF-2, Jun, Fos, or C/EBP whereas, ATF-2 will form heterodimers with Jun and Fos, but not with C/EBP or ATF-1. This strategy, therefore, allows a systematic approach to identifying, characterizing, and cloning proteins involved in the control of eukaryotic transcriptional regulation. The identification and characterization of the components of eukaryotic transcription complexes will allow studies that address the molecular mechanisms of normal and abnormal control of cellular gene expression.

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