Direct imaging of phosphorylation-dependent conformational change and DNA binding of CREB by electron microscopy.
BACKGROUND
The second messenger cAMP stimulates the expression of numerous genes through the PKA-dependent phosphorylation of CREB. The cAMP-regulated transcription factor CREB undergoes conformational change in response to phosphorylation by PKA at Ser 133. The phosphorylation enables interaction between the kinase-inducible domain (KID) of CREB and KIX domain of CREB binding protein (CBP).


RESULTS
To understand the activation mechanism of CREB-mediated gene expression, we performed the electron-microscope imaging of the transcription machinery. We improved the metal shadowing techniques to achieve higher resolution to detect phosphorylation-induced conformation change of the protein. Homodimer formation of CREB and the complex formation of phosphorylated CREB with CBP were observed under the electron microscope. The binding of the CREB dimer to CREs on the somatostatin and tyrosine hydroxylase promoters were also visualized directly and stereoscopically.


CONCLUSIONS
Greatly improved resolution achieved by our modified metal shadowing techniques makes it possible to visualize that the shape of CREB homodimer was changed in phosphorylation-dependent manner and that the promoter DNA strands containing CREs appeared to be bent and twisted slightly by the holding in the crevice of the CREB homodimer. This method may be applicable to visualize transcriptional activation process of nuclear receptors or general transcription machinery.
Usukura
J
J
Department of Anatomy, Nagoya University School of Medicine, Nagoya 466, Japan.
Nishizawa
Y
Y
Shimomura
A
A
Kobayashi
K
K
Nagatsu
T
T
Hagiwara
M
M
D000818:Animals; D050882:CREB-Binding Protein; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000648:ultrastructure*; D004247:DNA / Q000737:chemistry / Q000648:ultrastructure*; D019281:Dimerization; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013004:Somatostatin / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D013004:Somatostatin; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D050882:CREB-Binding Protein; C494365:Crebbp protein, rat