Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.     

Coordinate regulation of basal and cyclic 5'-adenosine monophosphate (cAMP)-activated expression of human chorionic gonadotropin-alpha by Ets-2 and cAMP-responsive element binding protein

Ets-2 controls the activities of many genes characteristically up-regulated in trophoblast. One apparent exception has been the gene for the human chorionic gonadotropin subunit alpha (hCGalpha). Here, we show that the hCGalpha gene contains two overlapping Ets binding sites adjacent to an activator protein-1-like site in its proximal promoter. Transactivation by Ets-2 is susceptible to truncation and mutation of these sites, which bind Ets-2 during in vitro mobility shift assays, as well as in vivo as determined by chromatin immunoprecipitation in choriocarcinoma cells. Knockdown of Ets-2 with short interfering RNA decreases both promoter activity and synthesis of hCGalpha. Ets-2 acts in combination with the protein kinase A (PKA) signal transduction pathway to activate the hCGalpha promoter expression. Mutation of the Ets-2 binding sites dramatically reduces up-regulation by PKA, whereas mutations within the two cAMP-responsive elements abolish responsiveness of the promoter to Ets-2. cAMP-responsive element binding protein (CREB) and Ets-2 form a complex that can be coimmunoprecipitated from choriocarcinoma cells, and association of CREB and Ets-2 is increased by activation of PKA. Regulation of hCGalpha subunit gene activity by cAMP involves the binding of CREB and Ets-2 to discrete elements in the promoter as well as a physical interaction between the two proteins. We propose that regulation of hCGalpha by Ets-2 and CREB enables coordinated expression of hCGalpha with its partner hCGbeta subunit.     

Ubiquitin-proteasome-mediated CREB repressor degradation during induction of long-term facilitation

Abstract Long-term facilitation in Aplysia and other forms of long-term memory in invertebrates and vertebrates require the gene expression cascade induced by cAMP-responsive element binding protein (CREB). Normally, gene expression by CREB is inhibited by repressors. The molecular mechanisms by which the repression is relieved are not understood. Our results show that Aplysia CREB repressor is a substrate for degradation by the ubiquitin-proteasome pathway. Treatment with the facilitatory neurotransmitter 5-hydroxy tryptamine (5-HT) leads to CREB repressor degradation in vivo and the degradation can be blocked by a specific proteasome inhibitor. Our biochemical studies show that attachment of ubiquitin molecules marks the CREB repressor for degradation by the proteasome. Protein kinase C (PKC) stimulates ubiquitination and degradation of the CREB repressor. Our results suggest that proteolytic removal of the CREB repressor is a potential mechanism for controlling gene expression by CREB. Without stimulation, gene expression is suppressed by the CREB repressor. Upon stimulation with 5-HT, PKC is activated, causing enhancement in ubiquitination and degradation of the CREB repressor. Thus, regulation of proteolysis of the CREB repressor by PKC might be critical in determining whether or not CREB-mediated gene expression goes forward during induction of long-term facilitation.