26Nature 1988 Aug 11;334(6182):494-8Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB.Yamamoto KK, Gonzalez GA, Biggs WH 3rd, Montminy MR.Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, CaliforniaA nuclear protein, CREB, has been isolated from rat brain and shown to stimulate transcription of the cyclic AMP-responsive gene somatostatin as a dimer. Biochemical analysis suggests that dimerization and transcriptional efficacy of CREB protein in vitro are regulated by phosphorylation. These findings demonstrate that cellular signals can modulate gene expression by regulating the covalent modification of pre-existing nuclear factors.
31Mechanism for generation of activators and repressors of cAMP-stimulated transcription
32CREB and CREM play roles in many physiological systems Memory and long-term potentiation(Silva et al., Annu. Rev. Neurosci. 21, )Circadian rhythms(Foulkes et al., Trends Neurosci. 20, )Pituitary function(Struthers et al., Nature 350, )Spermatogenesis(Sassone-Corsi P, Sem. Cell Dev. Biol. 9, )
33Aplysia californica (海兔) The function of the CREB has been modeled in transgenic organismsAplysia californica (海兔)"This sea-slug [Aplysia] is about five inches long; and is of a dirty-yellowish colour, veined with purple.... It feeds on the delicate seaweeds.... This slug, when disturbed, emits a very fine purplish-red fluid, which stains the water for the space of a foot around."CHARLES DARWIN, Voyage of the BeagleAplysia (genus Opisthobranchia) are hermaphrodites, meaning that both partners fertilize each others each with their sperm during mating.This gastropod mollusc is suited for Neurobiology mainly because of its large neurons - they are among the largest in the animal kingdom. In addition, its entire nervous system contains only a few hundred neurons, making it less complex and, hopefully, easier to understand than that of higher animals.Despite its simplicity, it exhibits a variety of behaviors and the capability to learn both non-associatively (sensitisation, habituation) and associatively (classical, operant conditioning).It was Aplysia that enabled researchers to study the molecular events in the cell during learning. In conjunction with the geneticly more powerful fly and mouse model systems, Aplysia research has boosted our understanding of the biological basis of learning and memory.Learning and memory stimulatedby serotonin as well as cAMP
34Drosophila as a model organism to study the role of CREB in learning and memoryFly (wt or mutants, obtained from wt fly fed with ethylmethane sulfonate, EMS)trained by odor avoidance learningBehaviorally tested in an odorant-associated electric shockCharacterize mutants with learning and memory deficiencydunce mutant: deficient in cAMP phosphodiesteraserutabaga mutant: mutation in adenylyl cyclase
354-methylcyclohexanol (MCH) n1 : n2 = ~50 : 50 for untrained flies Odor Avoidance Learning(one training cycle, 2.5 min total)100flies100flies100flies100flies100fliesElectrifiablecopper grid60 s45 s60 s45 s3-octanol (OCT) or4-methylcyclohexanol (MCH)(1o odor)Fresh airMCH or OCT(2o odor)Fresh airIn a T maze120 s forchoicen1n2OCTMCHn1 : n2 = ~50 : 50 for untrained flies
36Induction of a dominative CREB transgene specifically blocks long-term memory in DrosophilaYin et al., Cell 79, (1994)dCREB2:Drosophila CREB genedCREB2a:Activator for CREdCREB2b:Repressor for CREdCREB2-mLZ:Lost of Repressor function2 leucine in LZ domainvaline(dCREB-mLZ)25oC oC oCHS-induced dCREB2bcan be detected in brain cells
37Massed training cycle: 10 consecutive cycle withoutrest interval between themSpaced training cycle:10 consecutive cycle with a 15min rest period between eachwt flies+ 35 mM CXM, hr(before group)Training+ 35 mM CXM, 24 hr(after group)Odor avoidance exp.Performance Index (PI):100 means 100% avoidance of theshock-paired odor; 0 means a 50:50distribution in the T maze
38while 1 day memory after massed training and learning are normal Induction of the dCREB2-b transgene disrupts 1 day memory after spaced training,while 1 day memory after massed training and learning are normalCan-S: wt flies17-2 and M11-1: hs-dCREB2-btransgenic flies
39Induction of the mutant blocker (dCREB2-mLZ) does not affect 1 day memory after spaced training(dCREB2b)(dCREB2-mLZ)
40hs-dCREB2-b induction does not affect olfactory acuity or shock reactivity
41Induction of hs-dCREB2-b completely abolishes 7 day memory rentention
42( )The CREB and CREM transcription factors are activated by phosphorylation of a key serine residue by kinases stimulated by cyclic AMP, Ca2+, growth factors and stress signals. Phosphorylation allows recruitment of CREB binding protein (CBP), a large co-activator that contacts the general transcriptional machinery. Studies of the physiological roles played by CREB and CREM have uncovered novel routes of transcriptional activation. For example, in male germ cells CREM is not phosphorylated but associates with ACT, a member of the LIM-only class of proteins that has intrinsic transcriptional activity. Thus, in some circumstances, CREM can bypass the classical requirement for phosphorylation and association with CBP.