Abstract
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.
Highlights
JULY 23, 2010 VOLUME 285 NUMBER 30 like CREBbinding protein (CBP) or its paralogue P300 that bind to the kinase-inducible domain (KID) via the KID interaction domain (KIX) [10, 11]
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 cAMP-responsive element-binding protein (CREB)
All of these techniques are performed on dead cells and tissues, which allows conclusions on possible physiological events leading to CREB activation only to be made posthumously
Summary
Plasmid Construction—Amino acids 121–160 of the kinaseinducible domain (KID) and amino acids 586 – 662 of CREBbinding protein (CBP), including the linker GGSGGT, were generated by standard PCR from the full-length CREB cDNA. Both fragments were cloned into pRSET B (Invitrogen), the JOURNAL OF BIOLOGICAL CHEMISTRY 23285
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