Cooperative Mechanism of Transcriptional Activation by a Cyclic AMP-response Element Modulator α Mutant Containing a Motif for Constitutive Binding to CREB-binding Protein

Abstract

Cyclic AMP-response element modulator ␣ (CREM␣) is a transcription factor that is highly related to cAMPresponse element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREM␣ lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREM␣ to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREM␣ to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREM␣ DIEDML , constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREM␣ DIEDML and CREB DIEDML to direct their patterns of dimerization, we found that only CREM␣ DIEDML homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREM␣ DIEDML depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREM␣ DIEDML . Furthermore, a GAL4-C/EBP␣ fusion protein and CREM␣ DIEDML cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREM␣ to activate transcription despite its lack of Q regions.The cyclic AMP-response element modulator (CREM) 1 gene is alternatively spliced to generate multiple transcription factors that mediate the regulation of transcription by cAMP (reviewed in Ref. 1). These CREM factors form homodimers and heterodimers with the related protein CREB that bind specifically to CREs (TGACGTCA) via their conserved C-terminal basic leucine zipper (bZIP) domains (2). Three domains encoded by the CREM and CREB genes contribute to transcriptional activation: the kinase-inducible domain (KID) and two glutamine-rich regions designated as Q-1 and Q-2 (3). Phosphorylation of a serine residue within the KID by PKA allows CREB and CREM to bind to the coactivator CREB-binding protein (CBP) (4, 5). The Q regions are thought to interact with components of the basal transcriptional machinery (6). In CREB, the deletion of the Q-2 region prevents transcriptional activation (7). Moreover, CREM isoforms that lack Q regions have been shown to act as repressors of cAMP-induced transcription (2). These data have lead to the hypothesis that Q regions are required for CREB-mediated transcriptional activation.The CREM␣ isoform contains a KID but lacks both Q regions (2). CREM␣ represses transcription induced by PKA-activated CREB at a minimal promoter containing the somatostatin CRE (2). This repression may be mediated by CREB-CREM␣ heterodimers, which are only weak activators, or by CREM␣ homodimers that cannot activate transcription at the somatostatin CRE (8). These data form the bulk of the evidence for the hypot...

Insulin Regulation of PEPCK Gene Expression: A Model for Rapid and Reversible Modulation

“…However, it appears that cooperative effects are required for stable association in vivo [120], which is consistent with the observation that CREB alone mediates induction poorly [30,64]. Assuming that CBP is the primary co-regulator for cAMP induction of PEPCK, the requirement for cooperation in recruitment of CBP is consistent with the requirement for factors associated with both the CRE and the AC enhancer element to mediate induction [30][31][32][33]64].…”

Salt-inducible Kinase Represses cAMP-dependent Protein Kinase-mediated Activation of Human Cholesterol Side Chain Cleavage Cytochrome P450 Promoter through the CREB Basic Leucine Zipper Domain

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Cytokines increase CRE binding but decrease CRE-mediated reporter activity in rat hepatocytes by increasing c-Jun

“…pRC/RSV-CREB DIEDML is a mutant type of CREB expression plasmid in which Ser 133 region of wild-type CREB was replaced with DIEDML, which constitutively interacts with CREB binding protein (CBP). 10,11 Cell Treatment and Animal Surgical Procedure. Male Sprague-Dawley rats weighing 200 to 250 g (Harlan Sprague-Dawley, Madison, WI) were used in all experiments.…”