The cAMP response element binding protein CREB is a ubiquitously expressed transcription factor. It has been found to play a pivotal role in various physiological and developmental processes like learning and memory (Carlezon et al., 2005), glucose homeostasis (Herzig et al., 2003), cell survival (Mayr and Montminy, 2001) as well as neuronal development (Mantamadiotis et al., 2002). In accordance with that, CREB regulates the transcription of genes encoding for instance dopamine β-hydroxylase, somatostatin, glucagon, insulin, other transcription factors (e.g. c-Fos, Nurr1), or growth factors (e.g. brain-derived neurotrophic factor), among many others (Mayr and Montminy, 2001). Mantamadiotis and coworkers investigated effects of the loss of CREB on the development of the brain by generation of homozygous CREB-knockout (CREB-/-) mice.
CREB-/- mice died perinatally, but the loss of CREB was accompanied by an upregulation of the cAMP response element modulator (CREM), a member of the CREB family which potentially compensated in part for the loss of CREB. The conditional disruption of CREB- and CREM-function in the brain during development caused the perinatal death of mice due to generalized cell death in the nervous system. Strikingly, the postnatal knockout of CREB and CREM induced progressive neurodegeneration in the dorsolateral striatum as
well as in the CA1 and dentate gyrus region of the hippocampus (Mantamadiotis et al., 2002). These facts underline the importance of CREB for the development and maintenance of neuronal function.
3.a The structure of CREB
The initial cloning of the CREB cDNA revealed α-CREB and Δ-CREB, or CREB341 and CREB327, respectively. Resulting from alternative splicing of one of the eleven exons of the Creb gene the two forms differ in the presence of a 14 amino acid stretch termed the α-peptide (Figure 2), but are functionally equal. By alternative splicing of several 5’ exons the isoform β-CREB is generated: a protein lacking the first 40 amino acids and the α-peptide compared to α-CREB (Figure 2). All forms are expressed uniformly in human somatic cells (Lonze and Ginty, 2002; Shaywitz and Greenberg, 1999). The amino acid numbering in the present work refers to the Δ-CREB isoform.
CREB, as well as its family members activating transcription factor 1 (ATF1) and cAMP-response element modulator (CREM), belongs to the family of basic leucine zipper (bZip) transcription factors (Mayr and Montminy, 2001). The primary structure of CREB, as shown in Figure 2, demonstrates a centrally located 60 amino acid stretch referred to as the kinase inducible domain (KID). The hydrophobic glutamine-rich domains Q1 and Q2 flank the KID and are considered to be constitutively active. Dimerization of two CREB monomers is mediated by a conserved heptad repeat of leucine residues at the C-terminus called the leucine zipper. N-terminal to the leucine zipper, a lysine- and arginine-rich basic domain conveys the binding of CREB to DNA containing the octamer core sequence 5’-TGACGTCA-3’, the cAMP response element (CRE) (Lonze and Ginty, 2002;
Mayr and Montminy, 2001). By means of x-ray christallography the structure of the bZip bound to the CRE of the somatostatin-gene promoter was elucidated by Schumacher and colleagues in 2000. The analysis revealed the formation of a continuous α-helix by the bZip, in which the leucine zipper region forms a parallel coiled-coil interaction interface, and the basic region contacted the major groove of the DNA (Schumacher et al., 2000).
Interestingly, the crystal structure revealed a hexahydrated magnesium ion in the cavity between the bifurcating basic regions. The magnesium ion is located between the extended side-chains of the lysine-290 residues (K290) of the homodimer (Schumacher et al., 2000). The DNA binding of CREB strongly depends on the ability to coordinate that magnesium ion which was shown to be disrupted upon mutation of K290 (Craig et al., 2001; Dwarki et al., 1990; Schumacher et al., 2000).
Figure 2: Primary structure of CREB.
The Creb gene is composed of eleven exons (top). The α-CREB contains a C-terminal basic leucine zipper (bZip). The transactivation domain is composed of the central kinase inducible domain (KID) flanked by glutamine-rich domains Q1 and Q2, which are considered to be constitutively active (CAD). Upon alternative splicing Δ-CREB is generated lacking a 14 amino acid stretch termed α-peptide in the α-CREB isoform. Alternative splicing of several 5’ exons generates β-CREB lacking the first 40 amino acids of α-CREB and the α-peptide. The figure was modified from Lonze and Ginty, 2002.
3.b Transcriptional regulation mediated by CREB
The CREB-directed gene transcription is distinctly induced in response to numerous different signalling pathways. One critical trait is the phosphorylation of serine 119 (S119) situated in the KID (Mayr and Montminy, 2001). For instance, PKA phosphorylates CREB at S119 in response to elevated cAMP levels (Figure 4), and calcium/calmodulin-dependent kinases I, II and IV (CaMK I, II, and IV) phosphorylate S119 upon increased intracellular calcium levels after membrane depolarization (Figure 4). Furthermore, growth factors also lead to the phosphorylation of CREB by activation of pathways involving the family of mitogen-activated protein kinases (MAPK) resulting in the phosphorylation of S119 by the pp90 ribosomal S6 kinase family (RSKs) (Carlezon et al., 2005; Mayr and Montminy, 2001; Shaywitz and Greenberg, 1999). Essentially, the interaction with co-factors is a key feature of transcription regulation (Johannessen et al., 2004). CREB can interact with at least 30 other proteins affecting the CREB-directed gene transcription (Johannessen et al., 2004; McClung and Nestler, 2008). One of the best-described coactivators of CREB is the CREB binding protein (CBP) which is recruited to CREB in response to phosphorylation of S119. CBP possesses intrinsic histone deacetylase activity and is thought to promote CREB-directed transcription by association with RNA-polymerase II complexes (Mayr and Montminy, 2001; Shaywitz and Greenberg, 1999).
Additionally, CREB-directed gene transcription is promoted by interaction of the CREB-Q2
domain with TAFII130 of the TFIID complex belonging to the general transcriptional machinery (Nakajima et al., 1997).
Although the phosphorylation of CREB at S119 is believed to be necessary to activate CREB-directed gene-transcription, it is not sufficient. The immunosuppressive drugs cyclosporin A and FK506 potently block the stimulated CREB-directed gene transcription without interfering with the phosphorylation of S119 (Oetjen et al., 2005; Schwaninger et al., 1995; Schwaninger et al., 1993a). In 2003 Iourgenko et al. identified a new coactivator of CREB that associates to the bZip and promotes CREB-directed gene transcription independent of phosphorylation at S119. This new co-activator of CREB is termed transducer of regulated CREB (TORC) (Iourgenko et al., 2003).