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Direct observation of dimerization between different CREB1 isoforms in a living cell.

Sadamoto H, Saito K, Muto H, Kinjo M, Ito E - PLoS ONE (2011)

Bottom Line: In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor.Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells.As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan. sadamotoh@kph.bunri-u.ac.jp

ABSTRACT
Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

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Expression of fluorescent-labeled CREB1 isoform proteins in HeLa cells.A, Expression constructs. Each CREB1 activator (Act) or truncated activator (Act mut) proteins was fused with EGFP (green box), and each CREB1 repressor (Rep) or truncated repressor (Rep mut) protein was fused to tandem mRFP (red box). Within CREB1 isoform proteins, the functional domains are shown as patterned boxes. Schematic diagram of the fluorescent-labeled CREB1 isoform proteins are shown on the right side. B, Confocal microscopy images of HeLa cells transfected with expression constructs encoding EGFP- (green) or mRFP- (red) labeled CREB1 isoform proteins. CREB1 isoform proteins (EGFP-Act and mRFP-Rep, top) or truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut, bottom) were cotransfected, and the cells were imaged 16 h after transfection. Scale bars, 10 µm.
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pone-0020285-g001: Expression of fluorescent-labeled CREB1 isoform proteins in HeLa cells.A, Expression constructs. Each CREB1 activator (Act) or truncated activator (Act mut) proteins was fused with EGFP (green box), and each CREB1 repressor (Rep) or truncated repressor (Rep mut) protein was fused to tandem mRFP (red box). Within CREB1 isoform proteins, the functional domains are shown as patterned boxes. Schematic diagram of the fluorescent-labeled CREB1 isoform proteins are shown on the right side. B, Confocal microscopy images of HeLa cells transfected with expression constructs encoding EGFP- (green) or mRFP- (red) labeled CREB1 isoform proteins. CREB1 isoform proteins (EGFP-Act and mRFP-Rep, top) or truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut, bottom) were cotransfected, and the cells were imaged 16 h after transfection. Scale bars, 10 µm.

Mentions: We constructed four types of plasmids with two different fluorescent proteins for labeling: enhanced green fluorescent protein (EGFP) and tandem monomeric red fluorescent protein (mRFP) (Fig. 1A). Following a previous report [12], we used tandem mRFP to improve the weak brightness of mRFP. The CREB1 activator protein was fused to EGFP (EGFP-Act), whereas the CREB1 repressor protein was fused to mRFP (mRFP-Rep). Because the dimerization domains (bZIP domains) of the CREB1 isoform proteins exist at the C-terminal part, the expression constructs code for the N-terminal fusions of CREB1 activator or repressor isoform proteins with EGFP or tandem mRFP, respectively, to protect the dimerization ability of CREB1 isoform proteins. For negative control experiments, we also constructed plasmids encoding truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut). The dimerization domains of these proteins were selectively truncated without affecting the nucleus translocation signal (Fig. 1A).


Direct observation of dimerization between different CREB1 isoforms in a living cell.

Sadamoto H, Saito K, Muto H, Kinjo M, Ito E - PLoS ONE (2011)

Expression of fluorescent-labeled CREB1 isoform proteins in HeLa cells.A, Expression constructs. Each CREB1 activator (Act) or truncated activator (Act mut) proteins was fused with EGFP (green box), and each CREB1 repressor (Rep) or truncated repressor (Rep mut) protein was fused to tandem mRFP (red box). Within CREB1 isoform proteins, the functional domains are shown as patterned boxes. Schematic diagram of the fluorescent-labeled CREB1 isoform proteins are shown on the right side. B, Confocal microscopy images of HeLa cells transfected with expression constructs encoding EGFP- (green) or mRFP- (red) labeled CREB1 isoform proteins. CREB1 isoform proteins (EGFP-Act and mRFP-Rep, top) or truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut, bottom) were cotransfected, and the cells were imaged 16 h after transfection. Scale bars, 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105992&req=5

pone-0020285-g001: Expression of fluorescent-labeled CREB1 isoform proteins in HeLa cells.A, Expression constructs. Each CREB1 activator (Act) or truncated activator (Act mut) proteins was fused with EGFP (green box), and each CREB1 repressor (Rep) or truncated repressor (Rep mut) protein was fused to tandem mRFP (red box). Within CREB1 isoform proteins, the functional domains are shown as patterned boxes. Schematic diagram of the fluorescent-labeled CREB1 isoform proteins are shown on the right side. B, Confocal microscopy images of HeLa cells transfected with expression constructs encoding EGFP- (green) or mRFP- (red) labeled CREB1 isoform proteins. CREB1 isoform proteins (EGFP-Act and mRFP-Rep, top) or truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut, bottom) were cotransfected, and the cells were imaged 16 h after transfection. Scale bars, 10 µm.
Mentions: We constructed four types of plasmids with two different fluorescent proteins for labeling: enhanced green fluorescent protein (EGFP) and tandem monomeric red fluorescent protein (mRFP) (Fig. 1A). Following a previous report [12], we used tandem mRFP to improve the weak brightness of mRFP. The CREB1 activator protein was fused to EGFP (EGFP-Act), whereas the CREB1 repressor protein was fused to mRFP (mRFP-Rep). Because the dimerization domains (bZIP domains) of the CREB1 isoform proteins exist at the C-terminal part, the expression constructs code for the N-terminal fusions of CREB1 activator or repressor isoform proteins with EGFP or tandem mRFP, respectively, to protect the dimerization ability of CREB1 isoform proteins. For negative control experiments, we also constructed plasmids encoding truncated CREB1 proteins (EGFP-Act mut and mRFP-Rep mut). The dimerization domains of these proteins were selectively truncated without affecting the nucleus translocation signal (Fig. 1A).

Bottom Line: In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor.Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells.As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan. sadamotoh@kph.bunri-u.ac.jp

ABSTRACT
Cyclic AMP-responsive element binding protein 1 (CREB1) plays multiple functions as a transcription factor in gene regulation. CREB1 proteins are also known to be expressed in several spliced isoforms that act as transcriptional activators or repressors. The activator isoforms, possessing the functional domains for kinase induction and for interaction with other transcriptional regulators, act as transcriptional activators. On the other hand, some isoforms, lacking those functional domains, are reported to be repressors that make heterodimers with activator isoforms. The complex and ingenious function for CREB1 arises in part from the variation in their spliced isoforms, which allows them to interact with each other. To date, however, the dimerization between the activator and repressor isoforms has not yet been proved directly in living cells. In this study, we applied fluorescence cross-correlation spectroscopy (FCCS) to demonstrate direct observation of dimerization between CREB1 activator and repressor. The FCCS is a well established spectroscopic method to determine the interaction between the different fluorescent molecules in the aqueous condition. Using differently labeled CREB1 isoforms, we successfully observed the interaction of CREB1 activator and repressor via dimerization in the nuclei of cultured cells. As a result, we confirmed the formation of heterodimer between CREB1 activator and repressor isoforms in living cells.

Show MeSH
Related in: MedlinePlus