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. 2014 Jan 1;457(1):171-83.
doi: 10.1042/BJ20130963.

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

Xin-an Lu, Xiaofeng Wang, Wei Zhuo, Lin Jia, Yushan Jiang, Yan Fu, Yongzhang Luo

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

Xin-an Lu et al. Biochem J. .

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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Figures

Figure 1
Figure 1. Hsp90α interactions with PKCγ
(A) Lysates of HeLa cells transfected with control (Vector) or HA-tagged PKCγ-expressing vectors were subjected to immunoprecipitation (IP) with an anti-Hsp90α antibody (IP: Hsp90α), a control IgG (IgG) and an anti-HA antibody (HA). The immunoprecipitates were resolved by SDS/PAGE and immunoblotted with the respective antibodies. (B) Schematic diagram of Hsp90α fragments with a FLAG tag. (C) The expression level of FLAG-tagged Hsp90α fragments and HA-tagged PKCγ was detected after co-transfection. (D and E) The interaction of the WT N-terminal domain and middle domains of Hsp90α with PKCγ. Immunoprecipitates were immunoblotted with an anti-FLAG antibody to detect the immunoprecipitation efficiency and with an anti-HA antibody to detect the interacting domain of Hsp90α with PKCγ (D) and vice versa (E). CTD, C-terminal domain; IB, immunoblotting; MD, middle domain; NTD, N-terminal domain.
Figure 2
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.
Figure 3
Figure 3. PKCγ phosphorylation of Hsp90α
(A) Phosphorylation of Hsp90α by PKCγ at threonine and serine residues in vitro. pThr, phosphorylation of threonine; pSer, phosphorylation of serine; pSer-PKC-Substrate, phosphorylation of serine sites specifically by PKC. (B) Phosphorylation of Hsp90α by PKCγ in vivo after exogenous HA–PKCγ transfection into HeLa cells. (C) Phosphorylation of Hsp90α was detected upon the treatment with chelerythrine chloride (an inhibitor of PKC). (D) The phospho-threonine level of Hsp90α was detected after PKCγ knockdown. Upper panel, the knock-down efficacy was detected by Western blotting. Lower panel, the phospho-threonine level of Hsp90α was probed after immunoprecipitation. (E) The phospho-threonine level of Hsp90α was detected upon the treatment with PMA (an agonist of PKCγ). (F) The phospho-threonine level of Hsp90α was detected after different forms of PKCγ transfection. K380A, PKCγ with a K380A kinase-dead mutation; A25E, PKCγ with an A25E kinase open mutation; Ctrl, control; IB, immunoblotting; IP, immunoprecipitation.
Figure 4
Figure 4. Phosphorylation of Hsp90α Thr115, Thr425 and Thr603 by PKCγ
(A) Left-hand three lanes, threonine phosphorylation status of WT Hsp90α protein by PKCγ in vitro. Right-hand six lanes, threonine phosphorylation status of non-phospho-mimic Hsp90α mutants by PKCγ in vitro. (B) Threonine phosphorylation status of WT and non-phospho-mimics Hsp90α by PKCγ in vivo. HeLa cells transiently expressing Myc-tagged WT, T115A, T425A and T603A Hsp90α and HA-tagged PKCγ were lysed and ectopic Hsp90α proteins were immunoprecipitated with an anti-Myc antibody and immunoblotted with the indicated antibodies. (C) HeLa cells transfected with Myc-tagged WT Hsp90α or its non-phospho-mimics (single, double or triple site mutations) and HA-tagged PKCγ were lysed and immunoprecipitated (IP) with anti-Myc antibody and the immunoprecipitates were immunoblotted with an anti-phospho-Thr antibody. IB, immunoblotting; pThr, phosphorylation of threonine.
Figure 5
Figure 5. The effect of threonine phosphorylation of Hsp90α by PKCγ on Hsp90α chaperone function
(A) Binding of two forms of Hsp90α to high affinity ATP–agarose. WT Hsp90α was immunoprecipitated (IP) from control vector-transfected HeLa cells, whereas phospho-Thr-Hsp90α (pThr) was immunoprecipitated from HeLa cells transiently transfected with HA–PKCγ. (B) Binding of WT Hsp90α, T115A/E, T425A/E and T603A/E mutants to high affinity ATP–agarose. Proteins were ectopically expressed in HeLa cells and immunoprecipitated with anti-Myc antibodies. Supernatant was from the fraction after incubation with ATP–agarose. The pellet was ATP–agarose resolved with reducing SDS/PAGE loading buffer. (C) ATP calibration curve. The x axis show moles of ATP per assay and the y axis shows the relative light intensity. (D) Comparison of the ATPase activities of two endogenous Hsp90α proteins produced from HeLa cells transfected with pcDNA3.1 or HA-PKCγ. ***P<0.001. Results are means±S.D. (E) Comparison of ATPase activities of exogenous Hsp90α mutants produced from HeLa cells transfected with the indicated plasmids. ***P<0.001; *P<0.05; N.S, no significant difference. (F) HeLa cells co-transfected with control vector (Ctrl), Myc-tagged WT Hsp90α, T115A/E, T425A/E or T603A/E mutants, and HA-tagged Cdc37 were lysed and immunoprecipitated with an anti-Myc antibody. Co-precipitates were then detected by immunoblotting. (G) HeLa cells transfected with control vector, WT Hsp90α or T115A/E, T425A/E and T603A/E mutants were lysed and immunoprecipitated with an anti-Myc antibody. Co-precipitated endogenous (Endo) Hsp70 was then detected by immunoblotting. Exo, exogenous; IB, immunoblotting.
Figure 6
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).
Figure 7
Figure 7. The effects of PKCγ and Hsp90α on cancer cell migration and survival
(A and B) In the cell-migration assay, DMSO or 17-AAG was added to vector-, WT-PKCγ-, A25E-PKCγ- and K380A-PKCγ-transfected groups. After 6 h, migrated cells were examined (A) and quantified (B) (n=8). ***P<0.001; ###P<0.001; #P<0.05. Results are means±S.D. (C) HCT116 cell apoptosis detected by Western blotting of cleaved caspase 3.
Figure 8
Figure 8. A working model for reciprocal regulations of Hsp90α and PKCγ
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