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Interaction of Cx43 with Hsc70 regulates G1/S transition through CDK inhibitor p27.

Hino H, Dai P, Yoshida T, Hatakeyama T, Harada Y, Otsuji E, Okuda T, Takamatsu T - Sci Rep (2015)

Bottom Line: Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70.We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition.Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.

ABSTRACT
Connexin 43 (Cx43) functions as a cell growth suppressor. We have demonstrated that Cx43 interacts with heat shock cognate protein 70 (Hsc70) for regulating cell proliferation. Hsc70 interacts with CDK inhibitor p27, which regulates the assembly and subcellular localization of cyclin D1-CDK4-p27 complex. However, the involvement of p27 with Cx43-mediated cell cycle suppression is still poorly understood. Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70. We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition. Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

No MeSH data available.


Related in: MedlinePlus

Hsc70 regulates nuclear translocation of cyclin D1-CDK4-p27 complex.(a) Hsc70 did not affect the assembly of cyclin D1-CDK4-p27 complex. HuH-7 cells were transfected with equivalent plasmids of HA-p27, cMyc-cyclin D1and FLAG-CDK4 with or without T7-Hsc70. Lysates were immunoprecipitated by control IgG or anti-FLAG-tag antibody. The immunocomplexes were analyzed on 12% SDS-PAGE followed by western blotting (WB) using anti-FLAG-tag or anti-HA-tag, and/or anti-cMyc-tag, or anti-T7 tag antibodies. Cropped blots are shown. (b–d) Subcellular localization of the complex components CDK4 (b), cyclin D1 (c) and p27 (d). HuH-7 cells were transfected equivalent plasmids of HA-p27, cMyc-cyclin D1 and FLAG-CDK4 with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-FLAG-tag or anti-cMyc-tag, and/or anti-HA-tag antibodies was carried out, respectively. (e–g) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in CDK4 (e), cyclin D1 (f), and p27 (g). Nucleocytoplasmic localization analysis was performed in FLAG-tag, cMyc-tag and HA-tag, respectively. At least 30 transfected HuH-7 cells were examined and quantified for each sample. The data were plotted, and the horizontal lines represent median values. *p < 0.05. (h) A schematic illustration of the mechanism by which Cx43 regulates G1/S transition. Hcs70 interacts with the cyclin D1-CDK4-p27 complex and enhances the nuclear translocation of the complex, leading to G1/S transition (left). When Cx43 is up-regulated (right), the Cx43-Hsc70 interaction prevents the cyclin D1-CDK4-p27 complex enhancement for the nuclear translocation, causing inhibition of G1/S transition.
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f6: Hsc70 regulates nuclear translocation of cyclin D1-CDK4-p27 complex.(a) Hsc70 did not affect the assembly of cyclin D1-CDK4-p27 complex. HuH-7 cells were transfected with equivalent plasmids of HA-p27, cMyc-cyclin D1and FLAG-CDK4 with or without T7-Hsc70. Lysates were immunoprecipitated by control IgG or anti-FLAG-tag antibody. The immunocomplexes were analyzed on 12% SDS-PAGE followed by western blotting (WB) using anti-FLAG-tag or anti-HA-tag, and/or anti-cMyc-tag, or anti-T7 tag antibodies. Cropped blots are shown. (b–d) Subcellular localization of the complex components CDK4 (b), cyclin D1 (c) and p27 (d). HuH-7 cells were transfected equivalent plasmids of HA-p27, cMyc-cyclin D1 and FLAG-CDK4 with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-FLAG-tag or anti-cMyc-tag, and/or anti-HA-tag antibodies was carried out, respectively. (e–g) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in CDK4 (e), cyclin D1 (f), and p27 (g). Nucleocytoplasmic localization analysis was performed in FLAG-tag, cMyc-tag and HA-tag, respectively. At least 30 transfected HuH-7 cells were examined and quantified for each sample. The data were plotted, and the horizontal lines represent median values. *p < 0.05. (h) A schematic illustration of the mechanism by which Cx43 regulates G1/S transition. Hcs70 interacts with the cyclin D1-CDK4-p27 complex and enhances the nuclear translocation of the complex, leading to G1/S transition (left). When Cx43 is up-regulated (right), the Cx43-Hsc70 interaction prevents the cyclin D1-CDK4-p27 complex enhancement for the nuclear translocation, causing inhibition of G1/S transition.

Mentions: Next, to demonstrate whether the change in the amount of Hsc70 in cyclin D1-CDK4-p27 complex affects the subcellular distribution of the complex components of cyclin D1, CDK4 and p27, HuH-7 cells were co-transfected with cMyc-cyclin D1, HA-p27 and FLAG-CDK4 with or without T7-Hsc70. In co-immunoprecipitation assays, in spite of the presence or absence of Hsc70 in the complex, no changes of amounts of the complex components of CDK4, cyclin D1 and p27 were observed (Fig. 6a). However, compared with non-co-transfected with Hsc70 cells, significant increase of nuclear accumulation of the complex components of CDK4 (Fig. 6b,e), cyclin D1 (Fig. 6c,f), and p27 (Fig. 6d,g) were observed in those co-transfected with Hsc70 cells.


Interaction of Cx43 with Hsc70 regulates G1/S transition through CDK inhibitor p27.

Hino H, Dai P, Yoshida T, Hatakeyama T, Harada Y, Otsuji E, Okuda T, Takamatsu T - Sci Rep (2015)

Hsc70 regulates nuclear translocation of cyclin D1-CDK4-p27 complex.(a) Hsc70 did not affect the assembly of cyclin D1-CDK4-p27 complex. HuH-7 cells were transfected with equivalent plasmids of HA-p27, cMyc-cyclin D1and FLAG-CDK4 with or without T7-Hsc70. Lysates were immunoprecipitated by control IgG or anti-FLAG-tag antibody. The immunocomplexes were analyzed on 12% SDS-PAGE followed by western blotting (WB) using anti-FLAG-tag or anti-HA-tag, and/or anti-cMyc-tag, or anti-T7 tag antibodies. Cropped blots are shown. (b–d) Subcellular localization of the complex components CDK4 (b), cyclin D1 (c) and p27 (d). HuH-7 cells were transfected equivalent plasmids of HA-p27, cMyc-cyclin D1 and FLAG-CDK4 with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-FLAG-tag or anti-cMyc-tag, and/or anti-HA-tag antibodies was carried out, respectively. (e–g) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in CDK4 (e), cyclin D1 (f), and p27 (g). Nucleocytoplasmic localization analysis was performed in FLAG-tag, cMyc-tag and HA-tag, respectively. At least 30 transfected HuH-7 cells were examined and quantified for each sample. The data were plotted, and the horizontal lines represent median values. *p < 0.05. (h) A schematic illustration of the mechanism by which Cx43 regulates G1/S transition. Hcs70 interacts with the cyclin D1-CDK4-p27 complex and enhances the nuclear translocation of the complex, leading to G1/S transition (left). When Cx43 is up-regulated (right), the Cx43-Hsc70 interaction prevents the cyclin D1-CDK4-p27 complex enhancement for the nuclear translocation, causing inhibition of G1/S transition.
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Related In: Results  -  Collection

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Show All Figures
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f6: Hsc70 regulates nuclear translocation of cyclin D1-CDK4-p27 complex.(a) Hsc70 did not affect the assembly of cyclin D1-CDK4-p27 complex. HuH-7 cells were transfected with equivalent plasmids of HA-p27, cMyc-cyclin D1and FLAG-CDK4 with or without T7-Hsc70. Lysates were immunoprecipitated by control IgG or anti-FLAG-tag antibody. The immunocomplexes were analyzed on 12% SDS-PAGE followed by western blotting (WB) using anti-FLAG-tag or anti-HA-tag, and/or anti-cMyc-tag, or anti-T7 tag antibodies. Cropped blots are shown. (b–d) Subcellular localization of the complex components CDK4 (b), cyclin D1 (c) and p27 (d). HuH-7 cells were transfected equivalent plasmids of HA-p27, cMyc-cyclin D1 and FLAG-CDK4 with or without T7-Hsc70. After 48 hr of transfection, immunofluorescence staining with anti-FLAG-tag or anti-cMyc-tag, and/or anti-HA-tag antibodies was carried out, respectively. (e–g) Ratio of nuclear/cytoplasmic (N/C) fluorescence intensities in CDK4 (e), cyclin D1 (f), and p27 (g). Nucleocytoplasmic localization analysis was performed in FLAG-tag, cMyc-tag and HA-tag, respectively. At least 30 transfected HuH-7 cells were examined and quantified for each sample. The data were plotted, and the horizontal lines represent median values. *p < 0.05. (h) A schematic illustration of the mechanism by which Cx43 regulates G1/S transition. Hcs70 interacts with the cyclin D1-CDK4-p27 complex and enhances the nuclear translocation of the complex, leading to G1/S transition (left). When Cx43 is up-regulated (right), the Cx43-Hsc70 interaction prevents the cyclin D1-CDK4-p27 complex enhancement for the nuclear translocation, causing inhibition of G1/S transition.
Mentions: Next, to demonstrate whether the change in the amount of Hsc70 in cyclin D1-CDK4-p27 complex affects the subcellular distribution of the complex components of cyclin D1, CDK4 and p27, HuH-7 cells were co-transfected with cMyc-cyclin D1, HA-p27 and FLAG-CDK4 with or without T7-Hsc70. In co-immunoprecipitation assays, in spite of the presence or absence of Hsc70 in the complex, no changes of amounts of the complex components of CDK4, cyclin D1 and p27 were observed (Fig. 6a). However, compared with non-co-transfected with Hsc70 cells, significant increase of nuclear accumulation of the complex components of CDK4 (Fig. 6b,e), cyclin D1 (Fig. 6c,f), and p27 (Fig. 6d,g) were observed in those co-transfected with Hsc70 cells.

Bottom Line: Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70.We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition.Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.

ABSTRACT
Connexin 43 (Cx43) functions as a cell growth suppressor. We have demonstrated that Cx43 interacts with heat shock cognate protein 70 (Hsc70) for regulating cell proliferation. Hsc70 interacts with CDK inhibitor p27, which regulates the assembly and subcellular localization of cyclin D1-CDK4-p27 complex. However, the involvement of p27 with Cx43-mediated cell cycle suppression is still poorly understood. Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70. We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition. Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

No MeSH data available.


Related in: MedlinePlus