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The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.

Lu XA, Wang X, Zhuo W, Jia L, Jiang Y, Fu Y, Luo Y - Biochem. J. (2014)

Bottom Line: It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications.However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process.The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery.

View Article: PubMed Central - PubMed

ABSTRACT
It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr(115)/Thr(425)/Thr(603), of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a 'phosphorylation switch' for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

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PKCγ chaperoning by Hsp90α(A) Whole-cell lysates were prepared from control siRNA or Hsp90α siRNA (si-Hsp90α)-transfected HeLa cells. Protein levels of Hsp90α and PKCγ and the phospho-Thr514 level of PKCγ were then detected by their respective antibodies. N.C, negative control. (B) HeLa cells treated by 17-AAG in a dose-dependent manner for 12 h were prepared for SDS/PAGE and protein levels of phopho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were then detected by immunoblotting. (C) HeLa cells treated by 1 μM 17-AAG for different times were prepared for SDS/PAGE, and then protein levels of phospho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were detected by immunoblotting. (D) Protein levels of phospho-Thr514-PKCγ and PKCγ in different fractions of HeLa cells treated with 1 μM 17-AAG for 12 h were detected by Western blotting. GAPDH and Na+/K+-ATPase were used as loading controls for the cytosol and membrane fractions respectively. (E) HeLa cells were transfected with the control vector or HA–PKCγ, treated with 17-AAG at different concentrations for 12 h and then whole-cell lysates were immunoprecipitated (IP) with an anti-Hsp90α antibody. Co-immunoprecipitated exogenous HA-tagged PKCγ was detected by immunoblotting. (F) Hsp90α was immunoprecipitated with an anti-Hsp90α antibody from the cytosol and membrane compartments with/without 17-AAG treatment and then co-precipitated endogenous PKCγ was detected by immunoblotting. IB, immunoblotting.
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Figure 2: PKCγ chaperoning by Hsp90α(A) Whole-cell lysates were prepared from control siRNA or Hsp90α siRNA (si-Hsp90α)-transfected HeLa cells. Protein levels of Hsp90α and PKCγ and the phospho-Thr514 level of PKCγ were then detected by their respective antibodies. N.C, negative control. (B) HeLa cells treated by 17-AAG in a dose-dependent manner for 12 h were prepared for SDS/PAGE and protein levels of phopho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were then detected by immunoblotting. (C) HeLa cells treated by 1 μM 17-AAG for different times were prepared for SDS/PAGE, and then protein levels of phospho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were detected by immunoblotting. (D) Protein levels of phospho-Thr514-PKCγ and PKCγ in different fractions of HeLa cells treated with 1 μM 17-AAG for 12 h were detected by Western blotting. GAPDH and Na+/K+-ATPase were used as loading controls for the cytosol and membrane fractions respectively. (E) HeLa cells were transfected with the control vector or HA–PKCγ, treated with 17-AAG at different concentrations for 12 h and then whole-cell lysates were immunoprecipitated (IP) with an anti-Hsp90α antibody. Co-immunoprecipitated exogenous HA-tagged PKCγ was detected by immunoblotting. (F) Hsp90α was immunoprecipitated with an anti-Hsp90α antibody from the cytosol and membrane compartments with/without 17-AAG treatment and then co-precipitated endogenous PKCγ was detected by immunoblotting. IB, immunoblotting.

Mentions: To test whether the stability and activation status of PKCγ depends upon its interaction with Hsp90α, we knocked down Hsp90α expression in HeLa cells by siRNA and found that the abundance of overall PKCγ protein levels, as well as that of phospho-Thr514, were strikingly reduced compared with the controls (Figure 2A). We then confirmed these results by using a pharmacological inhibitor of Hsp90, 17-AAG, a derivative of geldanamycin [42]. After treating HeLa cells with 17-AAG, we measured the mRNA levels of PKCγ using qRT-PCR (Supplementary Figure S1 at http://www.biochemj.org/bj/457/bj4570171add.htm). Our data showed that the mRNA level of PKCγ was unchanged suggesting that the transcription level of PKCγ was not directly influenced by 17-AAG treatment. We then assayed the protein level and Thr514 phosphorylation status of PKCγ by Western blotting after 17-AAG treatment. We found the protein level of PKCγ and its phosphorylation at Thr514 in whole HeLa cell lysates were decreased in a time- and dose-dependent manner compared with the controls (Figures 2B and 2C). We then compared the effect of 17-AAG on PKCγ abundance in the membrane compared with the cytosol cell fractions. As shown in Figure 2(D), the PKCγ protein abundance was far more reduced in the membrane fraction than that in the cytosol fraction in 17-AAG-treated cells compared with the controls, indicating that the chaperone activity of Hsp90 is also very critical for PKCγ membrane translocation.


The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.

Lu XA, Wang X, Zhuo W, Jia L, Jiang Y, Fu Y, Luo Y - Biochem. J. (2014)

PKCγ chaperoning by Hsp90α(A) Whole-cell lysates were prepared from control siRNA or Hsp90α siRNA (si-Hsp90α)-transfected HeLa cells. Protein levels of Hsp90α and PKCγ and the phospho-Thr514 level of PKCγ were then detected by their respective antibodies. N.C, negative control. (B) HeLa cells treated by 17-AAG in a dose-dependent manner for 12 h were prepared for SDS/PAGE and protein levels of phopho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were then detected by immunoblotting. (C) HeLa cells treated by 1 μM 17-AAG for different times were prepared for SDS/PAGE, and then protein levels of phospho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were detected by immunoblotting. (D) Protein levels of phospho-Thr514-PKCγ and PKCγ in different fractions of HeLa cells treated with 1 μM 17-AAG for 12 h were detected by Western blotting. GAPDH and Na+/K+-ATPase were used as loading controls for the cytosol and membrane fractions respectively. (E) HeLa cells were transfected with the control vector or HA–PKCγ, treated with 17-AAG at different concentrations for 12 h and then whole-cell lysates were immunoprecipitated (IP) with an anti-Hsp90α antibody. Co-immunoprecipitated exogenous HA-tagged PKCγ was detected by immunoblotting. (F) Hsp90α was immunoprecipitated with an anti-Hsp90α antibody from the cytosol and membrane compartments with/without 17-AAG treatment and then co-precipitated endogenous PKCγ was detected by immunoblotting. IB, immunoblotting.
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Figure 2: PKCγ chaperoning by Hsp90α(A) Whole-cell lysates were prepared from control siRNA or Hsp90α siRNA (si-Hsp90α)-transfected HeLa cells. Protein levels of Hsp90α and PKCγ and the phospho-Thr514 level of PKCγ were then detected by their respective antibodies. N.C, negative control. (B) HeLa cells treated by 17-AAG in a dose-dependent manner for 12 h were prepared for SDS/PAGE and protein levels of phopho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were then detected by immunoblotting. (C) HeLa cells treated by 1 μM 17-AAG for different times were prepared for SDS/PAGE, and then protein levels of phospho-Thr514-PKCγ, PKCγ, Hsp90α and the loading control GAPDH were detected by immunoblotting. (D) Protein levels of phospho-Thr514-PKCγ and PKCγ in different fractions of HeLa cells treated with 1 μM 17-AAG for 12 h were detected by Western blotting. GAPDH and Na+/K+-ATPase were used as loading controls for the cytosol and membrane fractions respectively. (E) HeLa cells were transfected with the control vector or HA–PKCγ, treated with 17-AAG at different concentrations for 12 h and then whole-cell lysates were immunoprecipitated (IP) with an anti-Hsp90α antibody. Co-immunoprecipitated exogenous HA-tagged PKCγ was detected by immunoblotting. (F) Hsp90α was immunoprecipitated with an anti-Hsp90α antibody from the cytosol and membrane compartments with/without 17-AAG treatment and then co-precipitated endogenous PKCγ was detected by immunoblotting. IB, immunoblotting.
Mentions: To test whether the stability and activation status of PKCγ depends upon its interaction with Hsp90α, we knocked down Hsp90α expression in HeLa cells by siRNA and found that the abundance of overall PKCγ protein levels, as well as that of phospho-Thr514, were strikingly reduced compared with the controls (Figure 2A). We then confirmed these results by using a pharmacological inhibitor of Hsp90, 17-AAG, a derivative of geldanamycin [42]. After treating HeLa cells with 17-AAG, we measured the mRNA levels of PKCγ using qRT-PCR (Supplementary Figure S1 at http://www.biochemj.org/bj/457/bj4570171add.htm). Our data showed that the mRNA level of PKCγ was unchanged suggesting that the transcription level of PKCγ was not directly influenced by 17-AAG treatment. We then assayed the protein level and Thr514 phosphorylation status of PKCγ by Western blotting after 17-AAG treatment. We found the protein level of PKCγ and its phosphorylation at Thr514 in whole HeLa cell lysates were decreased in a time- and dose-dependent manner compared with the controls (Figures 2B and 2C). We then compared the effect of 17-AAG on PKCγ abundance in the membrane compared with the cytosol cell fractions. As shown in Figure 2(D), the PKCγ protein abundance was far more reduced in the membrane fraction than that in the cytosol fraction in 17-AAG-treated cells compared with the controls, indicating that the chaperone activity of Hsp90 is also very critical for PKCγ membrane translocation.

Bottom Line: It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications.However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process.The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery.

View Article: PubMed Central - PubMed

ABSTRACT
It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr(115)/Thr(425)/Thr(603), of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a 'phosphorylation switch' for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

Show MeSH
Related in: MedlinePlus