Imaging CREB Activation in Living Cells

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.Changes in gene expression are a hallmark of long term adaptive processes in many cell types and are crucially involved in the conversion from short term to long term plasticity of neuronal circuits. Transcription factors of the cAMP-responsive element-binding protein (CREB) 3 family have a prominent role in affecting such processes in various tissues and cell types (1-5). The family includes the homologous proteins CREB (6, 7), ATF-1 (8), and CREM (9). Upon stimulation a number of kinases phosphorylate the critical serine 133 residue within the kinase-inducible domain (KID) of CREB and the related transcription factors ATF-1 and CREM. Serine 133 phosphorylation leads to the recruitment of transcriptional coactivators, like CBP or its paralogue P300 that bind to the KID via the KID interaction domain (KIX) (10, 11). Interaction of CREB with CBP/P300 results in the assembly of the RNA-polymerase complex and in the expression of a large number of genes. Serine 133 phosphorylation is generally accepted as a key event in transcriptional regulation necessary although not in all cases sufficient, for CREB-mediated gene expression. Many studies focused on examining spatiotemporal profiles of CREB activation in cells and tissues by immunostainings using antibodies specific for serine 133 phosphorylation (for a selection, see Refs. 12-17). Few attempts were made to develop assays for real time imaging of CREB, a prerequisite for thorough analysis of the physiological events and the molecular dissection of signaling cascades converging on CREB (18,19). Genetically encoded indicators based on mutants of the green fluorescent protein (GFP) have become valuable tools to monitor spatiotemporal patterns of biochemical signals in live cells (20 -25). A number of indicators have ...

cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function

“…To improve the specificity of detecting CREB activation by PKGIγ residing in the nucleus, a phospho-CREB biosensor that is localized in the nucleus was employed for these studies. Previously, Friedrich and coworkers showed that a fusion protein consisting of the CREB kinase-inducible domain (KID), a linker, and the CREB-binding protein KID-interaction domain (KIX) sandwiched between an NLS and cyan fluorescent protein (CFP) on the NH 2 -terminal end and yellow fluorescence protein (YFP) on the COOH-terminal (referred to as NLS-ICAP ) exhibited reduced fluorescence resonance energy transfer (FRET) when PKA or calmodulin kinase induced CREB KID phosphorylation and KID-KIX interaction decreased CFP and YFP photonic interaction [35]). Because of the high variation in NLS-ICAP expression observed in the transfected cells during pilot studies, we first analyzed whether the activation of this phospho-CREB biosensor could be detected using fluorescence lifetime imaging microscopy (FLIM) instead of FRET.…”

“…Because cell fixation can affect the efficiency of protein fluorescence, we tested whether NLS-ICAP activation could be detected in formaldehyde-fixed cells using FLIM. In these studies, NLS-ICAP was modulated using forskolin because this had been shown to phosphorylate and modulate FRET of this CREB KID-based biosensor [35]. As shown in Fig.…”

“…5B indicate that the lack of CFP tm modulation by PKGIγ-ΔNLS was not due to a deficiency in PKGIγ-ΔNLS kinase activity; PKGIγ-ΔNLS was observed to phosphorylate over-expressed VASP, a PKGI phosphorylation target and member of the Ena/VASP family of proteins that resides in the cytosol, and to cause a known shift in its apparent molecular weight when over-expressed in the BHK cells. Because the NLS·ICAP CREB KID harbors several amino acid residues that are targeted by kinases that phosphorylate CREB [35, 48], we tested whether PKGIγ modulates the NLS-ICAP CFP tm through the consensus cyclic nucleotide-dependent phosphorylation site (-RRFS-, [49]). As shown in Fig.…”

“…The plasmids encoding NLS-ICAP and NLS-ICAP-ΔS 133 were a kind gift of Michael Friedrich [35], and NH 2 -terminal FLAG-epitope tagged vasodilator-stimulated phosphoprotein (FLAG-VASP) was from Michael Uhler [28]. pcDNA3-CFP was obtained from Addgene and pAcGFP1-Nuc, which encodes GFP-NLS, was purchased from Clontech.…”

Chemical biology toolkit for exploring protein kinase catalyzed phosphorylation reactions

“…Building on this principle, the imaging of the CREB phosphorylation was achieved in living cells by constructing the protein composed of the phosphorylated kinase-induced (KID) and the recognition domain (KIX) sandwitched between the FRET pair. Upon phosphorylation, the KID of CREB binds KIX domain to induce FRET 63. After transfection, the phosphorylation of the construct was comparable to the endogenous CREB phosphorylation and has allowed for measurements in living cells, due to the nontoxicity of the chimeric reporter.…”