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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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The effects of threonine set phosphorylation of Hsp90α on its interaction with PKCγ(A) Physical interaction between the HA–PKCγ protein and two forms of Hsp90α in vitro. HA–PKCγ immunoprecipitated (IP) from HeLa cells was transfected with HA–PKCγ. Control Hsp90α (Ctrl) was immunoprecipitated from HeLa cells transfected with a control vector. Phospho-Thr-Hsp90α (pT) was immunoprecipitated from HeLa cells transfected with Myc-tagged PKCγ. (B and C) HeLa cells co-transfected with HA–PKCγ and Myc-tagged Hsp90α non-phospho (B) or phospho (C) mutants were lysed and immunoprecipitated with an anti-Myc antibody. The co-precipitated exogenous HA–PKCγ was detected by immunoblotting (IB).
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Figure 6: The effects of threonine set phosphorylation of Hsp90α on its interaction with PKCγ(A) Physical interaction between the HA–PKCγ protein and two forms of Hsp90α in vitro. HA–PKCγ immunoprecipitated (IP) from HeLa cells was transfected with HA–PKCγ. Control Hsp90α (Ctrl) was immunoprecipitated from HeLa cells transfected with a control vector. Phospho-Thr-Hsp90α (pT) was immunoprecipitated from HeLa cells transfected with Myc-tagged PKCγ. (B and C) HeLa cells co-transfected with HA–PKCγ and Myc-tagged Hsp90α non-phospho (B) or phospho (C) mutants were lysed and immunoprecipitated with an anti-Myc antibody. The co-precipitated exogenous HA–PKCγ was detected by immunoblotting (IB).

Mentions: How matured kinase clients are released from the Hsp90 chaperone complex is a question of long-standing interest. Previous studies of the client-release process have mainly focused on the functions of Hsp90's intrinsic ATPase activity and co-chaperones such as Cdc37, Aha1 and PP5 (protein phosphatase 5). We proposed that the phosphorylation of Hsp90α by PKCγ can regulate the disassociation of PKCγ from its Hsp90α chaperone machinery. To prove this hypothesis, we immunoprecipitated WT and threonine residue-phosphorylated Hsp90α from HeLa cells transfected with control vector or overexpressing Myc–PKCγ and incubated these two forms of Hsp90α with the HA–PKCγ protein. As shown in Figure 6(A), the binding affinity between PKCγ and PKCγ-mediated threonine-phosphorylated Hsp90α was strikingly decreased compared with the WT Hsp90α. These data suggest that phosphorylation of Hsp90α, mediated by PKCγ, plays an important role in the disassociation of PKCγ from the Hsp90α chaperone machinery. To confirm further this conclusion, we explored the interactions between PKCγ and the Hsp90α non-phospho- and phospho-mimics. Although all of the Hsp90α non-phosphorylation mimics possessed a similar binding affinity for PKCγ (Figure 6B), the Hsp90α phosphorylation mimics showed increasingly lower binding affinities for PKCγ with an increasing number of mutated sites. Once all of the amino acids in the threonine residue set were converted into phosphorylation mimics, PKCγ completely lost its binding affinity to Hsp90α (Figure 6C). Taken together, these results demonstrate that PKCγ phosphorylation of chaperone Hsp90α triggers its own release from the Hsp90α chaperone machinery.


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)

The effects of threonine set phosphorylation of Hsp90α on its interaction with PKCγ(A) Physical interaction between the HA–PKCγ protein and two forms of Hsp90α in vitro. HA–PKCγ immunoprecipitated (IP) from HeLa cells was transfected with HA–PKCγ. Control Hsp90α (Ctrl) was immunoprecipitated from HeLa cells transfected with a control vector. Phospho-Thr-Hsp90α (pT) was immunoprecipitated from HeLa cells transfected with Myc-tagged PKCγ. (B and C) HeLa cells co-transfected with HA–PKCγ and Myc-tagged Hsp90α non-phospho (B) or phospho (C) mutants were lysed and immunoprecipitated with an anti-Myc antibody. The co-precipitated exogenous HA–PKCγ was detected by immunoblotting (IB).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927929&req=5

Figure 6: The effects of threonine set phosphorylation of Hsp90α on its interaction with PKCγ(A) Physical interaction between the HA–PKCγ protein and two forms of Hsp90α in vitro. HA–PKCγ immunoprecipitated (IP) from HeLa cells was transfected with HA–PKCγ. Control Hsp90α (Ctrl) was immunoprecipitated from HeLa cells transfected with a control vector. Phospho-Thr-Hsp90α (pT) was immunoprecipitated from HeLa cells transfected with Myc-tagged PKCγ. (B and C) HeLa cells co-transfected with HA–PKCγ and Myc-tagged Hsp90α non-phospho (B) or phospho (C) mutants were lysed and immunoprecipitated with an anti-Myc antibody. The co-precipitated exogenous HA–PKCγ was detected by immunoblotting (IB).
Mentions: How matured kinase clients are released from the Hsp90 chaperone complex is a question of long-standing interest. Previous studies of the client-release process have mainly focused on the functions of Hsp90's intrinsic ATPase activity and co-chaperones such as Cdc37, Aha1 and PP5 (protein phosphatase 5). We proposed that the phosphorylation of Hsp90α by PKCγ can regulate the disassociation of PKCγ from its Hsp90α chaperone machinery. To prove this hypothesis, we immunoprecipitated WT and threonine residue-phosphorylated Hsp90α from HeLa cells transfected with control vector or overexpressing Myc–PKCγ and incubated these two forms of Hsp90α with the HA–PKCγ protein. As shown in Figure 6(A), the binding affinity between PKCγ and PKCγ-mediated threonine-phosphorylated Hsp90α was strikingly decreased compared with the WT Hsp90α. These data suggest that phosphorylation of Hsp90α, mediated by PKCγ, plays an important role in the disassociation of PKCγ from the Hsp90α chaperone machinery. To confirm further this conclusion, we explored the interactions between PKCγ and the Hsp90α non-phospho- and phospho-mimics. Although all of the Hsp90α non-phosphorylation mimics possessed a similar binding affinity for PKCγ (Figure 6B), the Hsp90α phosphorylation mimics showed increasingly lower binding affinities for PKCγ with an increasing number of mutated sites. Once all of the amino acids in the threonine residue set were converted into phosphorylation mimics, PKCγ completely lost its binding affinity to Hsp90α (Figure 6C). Taken together, these results demonstrate that PKCγ phosphorylation of chaperone Hsp90α triggers its own release from the Hsp90α chaperone machinery.

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