title: Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge.
abstract: The cAMP response element binding protein CREB activates the transcription of genes in response to phosphorylation by cAMP-dependent protein kinase A (PKA) and other protein kinases. Phosphorylated CREB activates transcription by recruiting transcriptional co-activators such as the CREB binding protein. Here, we describe experiments that analyze the effects of phosphorylation on the DNA binding affinity of CREB and the structural characteristics of the CREB/DNA complex in solution. Analysis of deletion mutants of CREB indicate that amino acid sequences within the transactivation domain promote high-affinity binding of CREB to fluorescently labeled oligonucleotides containing cAMP response elements. In vitro experiments indicate that phosphorylation is processive between PKA as the initial kinase and glycogen synthase kinase-3 (GSK-3) but not casein kinase II as the secondary kinase. Fluorescent electrophoretic mobility shift assays show that phosphorylation by PKA results in a 3-5-fold increase in the binding affinity of CREB to both the symmetrical somatostatin CRE (SMS-CRE) and the asymmetric somatostatin upstream element (SMS-UE). Processive phosphorylation of CREB by GSK-3 attenuates the enhanced DNA binding in response to PKA thus acts as an inhibitor of PKA-induced binding. Ferguson plot analyses demonstrate that phosphorylation of CREB by PKA and GSK-3 result in an increase in the spherical size and the net positive surface charge of the CREB/DNA complex. Moreover, these analyses uncovered the unexpected finding that CREB associates as a tetramer both in the presence and absence of DNA. These findings suggest a model by which phosphorylation of CREB alters the secondary structure and charge characteristics of the CREB/DNA complex resulting in an alteration in binding affinity.
authors.affiliation: Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: Bullock
authors.forename: J F
authors.initials: JF
authors.lastname: Habener
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000502:physiology; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000502:physiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D047390:Casein Kinase II; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019793:Fluorescein; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D012045:Regulatory Sequences, Nucleic Acid; D013499:Surface Properties; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D000596:Amino Acids; D017362:Cyclic AMP Response Element-Binding Protein; D046911:Macromolecular Substances; D004247:DNA; D038341:Glycogen Synthase Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019793:Fluorescein