DREAM-αCREM Interaction via Leucine-Charged Domains Derepresses Downstream Regulatory Element-Dependent Transcription

Abstract

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, ␣CREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of ␣CREM is not required for the interaction, but phospho-␣CREM shows higher affinity for DREAM. The interaction with ␣CREM is independent of the Ca 2؉ -binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both ␣CREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca 2؉ .Transcriptional derepression is an important mechanism for the accurate control of gene expression. Transcriptional repressors can bind directly to DNA or act indirectly by interacting with other DNA-associated proteins (23,32). DREAM, a calcium-binding protein, represses basal expression of target genes through specific interaction with downstream regulatory elements (DREs) in the DNA (5, 6). Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca 2ϩ and protein kinase A (PKA) activation (5, 6). Other central players in the nuclear response to cyclic AMP (cAMP) and Ca 2ϩ are activator and repressor basic regionleucine zipper (LZ) transcription factors that bind to cAMPresponsive promoter elements (CREs) (10,15,25). They include proteins encoded by the CREB and CREM genes whose function is tightly regulated via phosphorylation by several kinases, including PKA and Ca 2ϩ -calmodulin-dependent kinases (8,12,13). As such, they represent the convergence point for various signaling cascades.The transcriptional repressor DREAM contains four EF hands, of which three (II, III, and IV) are responsible for the binding of calcium ions. In the absence of stimulated levels of nuclear calcium, DREAM binds with high affinity to the DRE sequence as a tetramer. Upon stimulation and increase in intracellular calcium, DREAM detaches from DNA without disruption of the tetrameric structure (6). The regulation by intracellular Ca 2ϩ of DREAM binding to DRE sites is a general mechanism that depends primarily on the EF-hand domains of DREAM. Mutation of two key amino acids within any of the functional EF hands results in mutated DREAM forms that stay bound to DNA also after calcium stimulation. Since DREAM binds to DRE sites as a tetramer, DREAM mutants insensitive to Ca 2ϩ behave as dominant negative mutants in a background of wild-type DREAM (unpublished observation). Similarly, PKA activation also results in loss of DREAM binding to the DRE and derepression of the target gene prodynorphin in human neuroblastoma cells (5). The molecular mechanism or the domains in DREAM that mediate this derepression by cAMP are unknown, and ...

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

“…24 The protein was purified and studied in the presence of LDAO since the previous study had shown that the addition of LDAO reduces protein aggregation and stabilizes apoDREAM and Ca 21 DREAM in its tetrameric and dimeric form, respectively. 24 We have observed that the presence of LDAO strongly impacts the emission spectra of Ca 21 free and Ca 21 bound DREAM. For example, DREAM samples dialyzed against 20 mM Tris buffer, 1 mM DTT, and 10 mM LDAO at pH 7.4 for 48 h provided emission spectra with a k max of 340 nm and 335 nm for apo-and Ca 21 DREAM, respectively, which are comparable to those reported by Osawa et al 24 On the other hand, emission spectra of DREAM samples prepared in the absence of LDAO are blue shifted with an emission maximum at $330 nm for Ca 21 21 association to DREAM leads to a conformational transition upon which the Trp residue moves towards the hydrophobic core of the protein.…”

“…EF-hand motif Ca 21 binding proteins are involved in the regulation of numerous intracellular processes ranging from gene expression, cell division, cell growth, as well as apoptosis. All members of this family carry at least one pair of EF-hand calcium binding sites that bind calcium with equilibrium affinity constants ranging from 10 4 M in the case of S100 proteins, to 10 8 M as found for parvalbumin.…”

“…In calmodulin, such highly dynamic protein structure enables specific interactions with a large number of intracellular targets. 2 Unlike the calmodulin family, structural changes linked to Ca 21 binding to EF-hands in S100 proteins are relatively modest and result in exposure of a wider and flatter hydrophobic crevice on the protein surface that facilitates binding of intracellular partners. 3 On the other hand, in neuronal calcium sensors (NCS), the calcium association leads to the structural reorganization that promotes repositioning of the N-and Cterminal domain and in some proteins exposure of the myristoyl group.…”

“…9 All members share a conserved region of 180 residues along with highly variable N-termini of 35 to 100 residues. 10 In DREAM, EF-hand 1 has no function due to the presence of Cys104 and Pro105 prevent this site from binding to Ca 21 /Mg 21 , whereas the canonical EF-hand 3 and EF-hand 4 functionally bind Ca 21 with a high affinity (K d $1-10 mM) and EF-hand 2 structurally binds Mg 21 (K d $14 mM). 26 KChIP1, DREAM, and KChIP4 are predominantly expressed in the brain where they regulate gating properties of Kv channels and the surface expression of the Kv4 complex.…”

“…20,21 The role of Ca 21 in regulating DREAM functions and the structural basis of DREAM interactions with multiple partners are not fully understood. Although the solution structure of Ca 21 bound DREAM (residues 76-256) is known 22 as well as the solution structure of the C-terminal domain (residues 161-256) of Ca 21 bound DREAM, 23 it is unclear how the Ca 21 / Mg 21 association to the EF hands alters the structural properties of DREAM. Considering the large number of DREAM interacting proteins, understanding the structural diversity could provide important insight into the relationship between the sequence, structure, and the target diversity of DREAM and NCS proteins in general.…”