Imaging CREB activation in living cells.
The Ca(2+)- and cAMP-responsive element-binding protein (CREB) and the related ATF-1 and CREM are stimulus-inducible transcription factors that link certain forms of cellular activity to changes in gene expression. They are attributed to complex integrative activation characteristics, but current biochemical technology does not allow dynamic imaging of CREB activation in single cells. Using fluorescence resonance energy transfer between mutants of green fluorescent protein we here develop a signal-optimized genetically encoded indicator that enables imaging activation of CREB due to phosphorylation of the critical serine 133. The indicator of CREB activation due to phosphorylation (ICAP) was used to investigate the role of the scaffold and anchoring protein AKAP79/150 in regulating signal pathways converging on CREB. We show that disruption of AKAP79/150-mediated protein kinase A anchoring or knock-down of AKAP150 dramatically reduces the ability of protein kinase A to activate CREB. In contrast, AKAP79/150 regulation of CREB via L-type channels may only have minor importance. ICAP allows dynamic and reversible imaging in living cells and may become useful in studying molecular components and cell-type specificity of activity-dependent gene expression.
Friedrich
Michael W
MW
Max-Planck-Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany.
Aramuni
Gayane
G
Mank
Marco
M
Mackinnon
Jonathan A G
JA
Griesbeck
Oliver
O
D054758:A Kinase Anchor Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D019703:Calcineurin / Q000378:metabolism; D002470:Cell Survival; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D015203:Reproducibility of Results
D054758:A Kinase Anchor Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin