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14-3-3θ is a binding partner of rat Eag1 potassium channels.

Hsu PH, Miaw SC, Chuang CC, Chang PY, Fu SJ, Jow GM, Chiu MM, Jeng CJ - PLoS ONE (2012)

Bottom Line: One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain.Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1.Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

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Phosphorylation-independent interaction of rEag1 with 14-3-3θ.(A) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. (Left panel) rEag1/rEag2 was co-expressed with an empty vector (−) or myc-tagged 14-3-3θ (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t-test: p<0.05).
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pone-0041203-g004: Phosphorylation-independent interaction of rEag1 with 14-3-3θ.(A) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. (Left panel) rEag1/rEag2 was co-expressed with an empty vector (−) or myc-tagged 14-3-3θ (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t-test: p<0.05).

Mentions: To further confirm the foregoing GST pull-down results, we tested the association of 14-3-3θ with full-length rEag1 by transiently co-expressing myc-14-3-3θ and rEag1 proteins in HEK293T cells. As shown in the left panel of Figure 4A, upon immunoprecipitating myc-14-3-3θ with the anti-myc antibody, a significant rEag1 protein signal was detected on the immunoblot. Conversely, myc-14-3-3θ was also immunoprecipitated with the anti-rEag1 antibody (Fig. 4A, right panel), indicating that rEag1 indeed co-existed in the same protein complex with 14-3-3θ. By contrast, no significant co-immunoprecipitation pattern was found for 14-3-3θ and rEag2, an isoform of rEag1 (Fig. 4A, left panel).


14-3-3θ is a binding partner of rat Eag1 potassium channels.

Hsu PH, Miaw SC, Chuang CC, Chang PY, Fu SJ, Jow GM, Chiu MM, Jeng CJ - PLoS ONE (2012)

Phosphorylation-independent interaction of rEag1 with 14-3-3θ.(A) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. (Left panel) rEag1/rEag2 was co-expressed with an empty vector (−) or myc-tagged 14-3-3θ (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t-test: p<0.05).
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pone-0041203-g004: Phosphorylation-independent interaction of rEag1 with 14-3-3θ.(A) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. (Left panel) rEag1/rEag2 was co-expressed with an empty vector (−) or myc-tagged 14-3-3θ (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t-test: p<0.05).
Mentions: To further confirm the foregoing GST pull-down results, we tested the association of 14-3-3θ with full-length rEag1 by transiently co-expressing myc-14-3-3θ and rEag1 proteins in HEK293T cells. As shown in the left panel of Figure 4A, upon immunoprecipitating myc-14-3-3θ with the anti-myc antibody, a significant rEag1 protein signal was detected on the immunoblot. Conversely, myc-14-3-3θ was also immunoprecipitated with the anti-rEag1 antibody (Fig. 4A, right panel), indicating that rEag1 indeed co-existed in the same protein complex with 14-3-3θ. By contrast, no significant co-immunoprecipitation pattern was found for 14-3-3θ and rEag2, an isoform of rEag1 (Fig. 4A, left panel).

Bottom Line: One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain.Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1.Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

ABSTRACT
The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.

Show MeSH
Related in: MedlinePlus