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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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FCCS measurement of differently labeled CREB1 isoforms in the nuclei of living cells.The inset is a schematic diagram showing fluorescent-labeled proteins used with each experiment. Auto- and cross-correlation curves of fluorescent-labeled proteins were shown for EGFP- mRFP chimera protein (A), EGFP-Act and mRFP-Rep (B), EGFP-Act mut and mRFP-Rep mut(C), and EGFP-Act and mRFP-Rep mut (D), respectively. The green curve denotes the auto-correlation of the green channel [Gg(τ)], the red curve the auto-correlation in the red channel [Gr(τ)] and the black curve the cross-correlation curve [Gc(τ)].
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pone-0020285-g002: FCCS measurement of differently labeled CREB1 isoforms in the nuclei of living cells.The inset is a schematic diagram showing fluorescent-labeled proteins used with each experiment. Auto- and cross-correlation curves of fluorescent-labeled proteins were shown for EGFP- mRFP chimera protein (A), EGFP-Act and mRFP-Rep (B), EGFP-Act mut and mRFP-Rep mut(C), and EGFP-Act and mRFP-Rep mut (D), respectively. The green curve denotes the auto-correlation of the green channel [Gg(τ)], the red curve the auto-correlation in the red channel [Gr(τ)] and the black curve the cross-correlation curve [Gc(τ)].

Mentions: To investigate the molecular interactions between CREB1 activator and repressor isoform proteins, we applied dual-color fluorescence cross-correlation spectroscopy (FCCS) to living cells. Typical auto-correlation curves and cross-correlation curve of FCCS data were shown in Figure 2. As a positive control, we measured the cross-correlation for the interaction between EGFP and mRFP fluorescent molecules using EGFP-mRFP fusion proteins (EGFP-mRFP chimera, Fig. 2A). The interaction between different CREB1 isoforms was also examined using fluorescent-labeled CREB1 activator and repressor proteins (EGFP-Act and mRFP-Rep, Fig. 2B). For one of the negative control experiments, we applied CREB1 isoforms lacking the dimerization domains (EGFP-Act mut and mRFP-Rep mut, Fig. 2C). Further, to ensure that the dimerization domain was not responsible for nonspecific interaction between CREB1 isoforms, we also performed another negative control experiment using EGFP-labeled CREB1 activator (EGFP-Act) that possesses the ability of dimerization (EGFP-Act and mRFP-Rep mut, Fig. 2D). For each measurement, we concerned to keep the ratio of expressed proteins constant to accurately examine the interaction between EGFP-labeled and mRFP-labeled proteins. For the positive control, EGFP and tandem mRFP fusion protein (EGFP-mRFP chimera) were used and the fluorescent intensity at the red channel of tandem mRFP was about 3-fold that of EGFP. Thus, during FCCS analysis, we chose the cells having the roughly the same green and red fluorescent intensity ratios as the EGFP-mRFP chimera (the ratio of count rate for red/green: 2.6–3.4). The cross-correlation for the negative control measurement could be considered as the background, however, it could be attributable to the leakage of one fluorescence emission through another fluorescence detector as described previously [15]. The uniform ratio between red/green signals can correct the effect of the fluorescence leakage from another channel on the cross-correlation.


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)

FCCS measurement of differently labeled CREB1 isoforms in the nuclei of living cells.The inset is a schematic diagram showing fluorescent-labeled proteins used with each experiment. Auto- and cross-correlation curves of fluorescent-labeled proteins were shown for EGFP- mRFP chimera protein (A), EGFP-Act and mRFP-Rep (B), EGFP-Act mut and mRFP-Rep mut(C), and EGFP-Act and mRFP-Rep mut (D), respectively. The green curve denotes the auto-correlation of the green channel [Gg(τ)], the red curve the auto-correlation in the red channel [Gr(τ)] and the black curve the cross-correlation curve [Gc(τ)].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105992&req=5

pone-0020285-g002: FCCS measurement of differently labeled CREB1 isoforms in the nuclei of living cells.The inset is a schematic diagram showing fluorescent-labeled proteins used with each experiment. Auto- and cross-correlation curves of fluorescent-labeled proteins were shown for EGFP- mRFP chimera protein (A), EGFP-Act and mRFP-Rep (B), EGFP-Act mut and mRFP-Rep mut(C), and EGFP-Act and mRFP-Rep mut (D), respectively. The green curve denotes the auto-correlation of the green channel [Gg(τ)], the red curve the auto-correlation in the red channel [Gr(τ)] and the black curve the cross-correlation curve [Gc(τ)].
Mentions: To investigate the molecular interactions between CREB1 activator and repressor isoform proteins, we applied dual-color fluorescence cross-correlation spectroscopy (FCCS) to living cells. Typical auto-correlation curves and cross-correlation curve of FCCS data were shown in Figure 2. As a positive control, we measured the cross-correlation for the interaction between EGFP and mRFP fluorescent molecules using EGFP-mRFP fusion proteins (EGFP-mRFP chimera, Fig. 2A). The interaction between different CREB1 isoforms was also examined using fluorescent-labeled CREB1 activator and repressor proteins (EGFP-Act and mRFP-Rep, Fig. 2B). For one of the negative control experiments, we applied CREB1 isoforms lacking the dimerization domains (EGFP-Act mut and mRFP-Rep mut, Fig. 2C). Further, to ensure that the dimerization domain was not responsible for nonspecific interaction between CREB1 isoforms, we also performed another negative control experiment using EGFP-labeled CREB1 activator (EGFP-Act) that possesses the ability of dimerization (EGFP-Act and mRFP-Rep mut, Fig. 2D). For each measurement, we concerned to keep the ratio of expressed proteins constant to accurately examine the interaction between EGFP-labeled and mRFP-labeled proteins. For the positive control, EGFP and tandem mRFP fusion protein (EGFP-mRFP chimera) were used and the fluorescent intensity at the red channel of tandem mRFP was about 3-fold that of EGFP. Thus, during FCCS analysis, we chose the cells having the roughly the same green and red fluorescent intensity ratios as the EGFP-mRFP chimera (the ratio of count rate for red/green: 2.6–3.4). The cross-correlation for the negative control measurement could be considered as the background, however, it could be attributable to the leakage of one fluorescence emission through another fluorescence detector as described previously [15]. The uniform ratio between red/green signals can correct the effect of the fluorescence leakage from another channel on the cross-correlation.

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