Kit Contents:

Component	Specification	Concentration (total volume)
CRE Reporter	A mixture of inducible CREB-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

General performance

Average maximum response rate = 213.5
Average Z' factor at maximum response rate = 0.77
Average coefficient of variation (CV%) = 7.6%

Excellent signal to noise ratio and dose dependent response

Cignal CRE reporter assay showed dose dependent increase in the activity of cAMP pathway: 293-H cells were transfected with CRE reporter and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, cells were treated with different doses of forskolin for 6 hours. (Forskolin increases the intracellular level of cAMP). Dual Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal CRE reporter assay measured 213 fold increase in the transcription activity of CRE-binding protein (CREB) and, in turn, in the activity of corresponding cAMP signaling pathway by 10µM forskolin.

The process of consolidating a new memory and the dynamic complexity of information processing within neuronal networks is greatly increased by activity-dependent changes in gene expression within individual neurons. A leading paradigm of such regulation is the activation of the nuclear transcription factor CREB (cAMP Responsive Element Binding Protein), and its family members the ATF (Activating Transcription Factor) and CREM (cAMP Response Element Modulator), which belong to bZIP (basic/leucine zipper) class of transcription factors that functions in vivo to regulate the proliferation of pituitary cells and thymocytes. Proteins belonging to this class are characterized by the ability to bind to the consensus sequence TGACGTCA (Ref.1, 2 & 3) and contain a leucine zipper responsible for DNA binding (basic region) and for dimerization (leucine zipper region) of the proteins. CREB can form homodimers or heterodimers with other members of the ATF family, including ATF1 and CREM. However, heterodimerization of CREB with other members of the ATF family decreases its stability and CRE (cAMP Responsive Element) binding affinity (Ref.4).

Changing levels of cAMP, Ca2+ and TGF-Beta (Transforming Growth Factor-Beta) regulate CREB and its closely related proteins (SHC, GRB2, SOS, HRas, cRaf, etc) that implicate in a variety of biological responses such as neuronal excitation, long-term memory formation, neural cell proliferation, and opiate tolerance (Ref.5). Through interaction with its nuclear partner CBP (CREB Binding Protein), it drives the transcription of a large number of genes. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. The crucial event in the activation of CREB is the phosphorylation of Ser133 in KID (Kinase-Inducible Domain). This domain includes several consensus phosphorylation sites for a variety of kinases like PKA (Protein Kinase-A), PKC (Protein Kinase-C), CSNK (Casein Kinases), CaMKs (Calmodulin Kinases), GSK3 (Glycogen Synthase Kinase-3) and p70S6K that can either increase or decrease the activity of CREB. Ser133 phosphorylation of CREB can be caused by electrical activity, Growth Factors, Neurotransmitter or Hormone action on GPCR (G-Protein-Coupled Receptors), or by Neurotrophin effects on RTKs (Receptor Tyrosine Kinases) (Ref.6). Upon stimulation of cellular GPCR (G-Protein-Coupled Receptors) and Growth Factor Receptors, AC (Adenylate Cyclase) is activated, by G-proteins: GN-Alpha, GN-Beta and GN-Gamma leading to increases in cAMP. This in turn activates PKA by dissociating the regulatory (PKAR) from the catalytic (PKAC) subunits. In the basal state, PKA resides in the cytoplasm as an inactive heterotetramer of paired regulatory and catalytic subunits. Induction of cAMP liberates the catalytic subunits. This activated PKAC then recruits the Ca2+/CalmK-IV (Calmodulin (Calm)-dependent Kinases), MEK (MAPK/ERK Kinases)/ ERK1/2 (Extracellular Signal-Regulated Kinases) and together they translocate to the nucleus (Ref.7 & 11). In the nucleus they lead to the recruitment of the transcriptional coactivators CBP (CREB Binding Protein) and p300 by phosphorylating Elk1. Elk1 is a part of a TCF (Ternary Complex Factor) that activates RSKs (Ribosomal S6 Kinases) and binds SRF (Serum Response Factor) to the SRE (Serum Response Element). Phosphorylation of Elk1 increases its transcriptional ability to form ternary complexes with SRF at the SRE in the promoter region of many genes, such as c-Fos (Ref.8). CBP/p300 stimulates gene expression by interacting with components of the general transcriptional machinery or by promoting the acetylation of specific lysine residues in nucleosomes located near transcriptionally active promoters thus creating access to the gene for the basal transcriptional machinery. The basal transcriptional machinery includes TBP (TATA-binding protein), TFIIB (Transcription Factor-II-B), and RNA Pol-II (RNA Polymerase-II) (Ref.9). The accumulation of cAMP in response to activation of GPCR also induces PLC-Gamma (Phospholipase-C-Gamma) that catalyzes the formation of DAG (Diacylglycerol), a PKC activator through PI (Phosphatidylinositols). PI3K (Phosphoinositide-3kinase) is responsible for activation of Akt/PKB (Protein Kinase-B) which directly or indirectly affects CREB.

In the presynaptic terminal, GLUR (metabotropic Glutamate Receptors Group-I) augment Glu (Glutamate) release via interaction of PKC and PKA whereas Group-II/III Receptors inhibit Glutamate release. Phosphorylation of Group-II/III Receptors (metabotropic Glutamate Receptors Group-II/III) also inhibits the transmitter release. These activities can indirectly regulate CREB and Elk1 phosphorylation in the postsynaptic neurons. In the postsynaptic striatal neurons, Group-I Receptors increase PKC activity as well as intracellular Ca2+ levels from internal store via PLC/DAG and PI/IP3 Pathways, respectively. Activated PKC induces an increase in extracellular Ca2+ influx through phosphorylation of iGluR (ionotropic Glutamate Receptors), in particular NMDARs (N-Methyl-D-Aspartate Receptors). Elevation of Ca2+ through CaCn (Calcium Channel) upregulates Ca2+-dependent CaMK-II/ ERK1/2 signaling cascades resulting in CREB and Elk1 phosphorylation. In contrast, Group-II/III Receptors suppress the Ca2+ cascades by inhibiting AC coupling to GPCRs such as Dopamine Receptors. The decreased cAMP level reduces PKA-dependent phosphorylation of NMDARs (Ref.10).

CREB can be phosphorylated at a number of sites other than Ser133, including Ser129, Ser142 and Ser143. Phosphorylation of Ser142 and dephosphorylation of Ser133 residue by CalmK represses CREB activity. Calcineurin dependent PP1 and PP2A (Protein Phosphatases) is involved in the dephosphorylation of CREB. In addition to dephosphorylation, repressors can also block CREB activity (Ref.9). The activation of plasma-membrane channels, including NMDARs and L-VGCCs also relieve repressor factors such as DREAM (Downstream Response Element (DRE)-Antagonist Modulator), and induce other activators like SRF, that work with CREB to drive c-Fos transcription. By contrast, only stimuli that elevate intracellular Ca2+ (by NMDAR and L-VGCC activation) lead to the phosphorylation of CREB at Ser142 and Ser143, and activate the transcription factor CaRF (Calcium Response Factor). Phosphorylation of Ser142/143 in CREB inhibits the association of phosphorylated Ser133 with CBP. CaRF cooperates with CREB to promote transcription of BDNF (Brain-Derived Neurotrophic Factor), assisting in cofactor recruitment and mediation of stimulus-selective gene transcription (Ref.8).

The cAMP/CREB signaling pathway has been strongly implicated in the regulation of a wide range of biological functions such as growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms and the synaptic plasticity that is associated with a variety of complex forms of memory including spatial and social learning indicating that CREB may be a universal modulator of processes required for memory formation (Ref.6). Deletion of CREB and CREM in neurons of the developing CNS (Central Nervous System) results in apoptosis, and postnatal ablation of these genes results in neuronal degeneration in adulthood. Neurons of the adult striatum and hippocampus are particularly vulnerable to CREB/CREM deficiency. The richness of CREB signaling is greatly increased by its responsiveness to multiple intracellular signal transduction cascades and the potential for this family of transcription factors to induce and suppress gene expression renders them ideally suited for regulating gene expression during the process of epidermal differentiation (Ref.4).

References:

1. Josselyn SA, Nguyen PV.
CREB, synapses and memory disorders: past progress and future challenges.
Curr. Drug Targets CNS Neurol. Disord. 2005 Oct; 4(5):481-97
PubMed ID: 16266283

2. Carlezon WA Jr, Duman RS, Nestler EJ.
The many faces of CREB.
Trends. Neurosci. 2005 Aug; 28(8):436-45.
PubMed ID: 15982754

3. Dolmetsch RE, Pajvani U, Fife K, Spotts JM, Greenberg ME.
Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway.
Science. 2001 Oct 12; 294(5541): 333-9.
PubMed ID: 11598293

4. Johannessen M, Delghandi MP, Seternes OM, Johansen B, Moens U.
Synergistic activation of CREB-mediated transcription by forskolin and phorbol ester requires PKC and depends on the glutamine-rich Q2 transactivation domain.
Cell Signal. 2004 Oct; 16(10):1187-99.
PubMed ID: 15240013

5. Wu GY, Deisseroth K, Tsien RW.
Activity-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway.
Proc. Natl. Acad. Sci. U S A. 2001 Feb 27; 98(5): 2808-13.
PubMed ID: 11226322

6. Mayr B, Montminy M.
Transcriptional regulation by the phosphorylation-dependent factor CREB.
Nat. Rev. Mol. Cell Biol. 2001 Aug; 2(8): 599-609.
PubMed ID: 11483993

7. Choe ES, Wang JQ.
Regulation of transcription factor phosphorylation by metabotropic glutamate receptor-associated signaling pathways in rat striatal neurons.
Neuroscience. 2002; 114(3): 557-65.
PubMed ID: 12220559

8. Finkbeiner S.
New roles for introns: sites of combinatorial regulation of Ca2+- and cyclic AMP-dependent gene transcription.
Sci. STKE. 2001 Aug 7; 2001(94): PE1.
PubMed ID: 11752669

9. Chawla S, Bading H.
CREB/CBP and SRE-interacting transcriptional regulators are fast on-off switches: duration of calcium transients specifies the magnitude of transcriptional responses.
J. Neurochem. 2001 Nov; 79(4):849-58.
PubMed ID: 11723177

10. Boulware MI, Weick JP, Becklund BR, Kuo SP, Groth RD, Mermelstein PG.
Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein.
J. Neurosci. 2005 May 18; 25(20):5066-78.
PubMed ID: 15901789

11. Ahmed T, Frey JU.
Plasticity-specific phosphorylation of CaMKII, MAP-kinases and CREB during late-LTP in rat hippocampal slices in vitro.
Neuropharmacology. 2005 Sep; 49(4):477-92.
PubMed ID: 16005911



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