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. 2008 Sep 26;31(6):886-95.
doi: 10.1016/j.molcel.2008.07.021.

Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37

Cara K Vaughan  1 , Mehdi Mollapour, Jennifer R Smith, Andrew Truman, Bin Hu, Valerie M Good, Barry Panaretou, Len Neckers, Paul A Clarke, Paul Workman, Peter W Piper, Chrisostomos Prodromou, Laurence H Pearl
Affiliations

Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37

Cara K Vaughan et al. Mol Cell. .

Abstract

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

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Figures

Figure 1
Figure 1
Specificity of the Polyclonal Anti-PhosphoSer13-Cdc37 Antibody (A) Coomasie-stained SDS-PAGE of purified H-C-K and C-K complexes. (B) Incubation of purified WT-Cdc37 expressed in E. coli with CK2 at 30°C shows a time-dependent specific signal from the α-pSer13-Cdc37 polyclonal antibody. No signal was observed with an S13A mutant. The blot was amido black-stained as a loading control. It has been shown elsewhere that Ser13 is the only site of phosphorylation by CK2 on Cdc37. (C) Free Cdc37, Cdc37 in complex with Cdk4 (C-K) and Cdc37 in complex with Hsp90 and Cdk4 (H-C-K) expressed in insect cells are all phosphorylated on Ser13.
Figure 2
Figure 2
Dephosphorylation of Free Cdc37 or Cdc37 within the C-K and H-C-K Complexes by λPP (A) The rate of dephosphorylation of free Cdc37, the C-K complex, and the H-C-K complex were compared. Samples with approximately equivalent amounts of Cdc37 were incubated in the absence (−) and presence (+) of λPP at room temperature for the specified times. Western blots were probed with α-pSer13-Cdc37. The blots were stripped and reprobed with α-Cdc37 as a loading control. (B) The C-K complex was incubated in the absence (−) and presence (+) of λPP and the effect of dephosphorylation on composition examined by analytical gel filtration with a Superose 6 10/300 GL column; 1 ml fractions were probed for each component of the complex by Western blot. Dephosphorylation was confirmed by probing with α-pSer13-Cdc37. Cdk4 alone elutes in fraction 19 (data not shown). (C) The H-C-K complex was incubated in the absence (−) or presence (+) of λPP. At specified time points, samples were withdrawn and the effect of dephosphorylation on complex stability assessed by pulldown of the complex via the His6-tagged Cdk4 on Talon resin. Samples were probed by Western blot for Cdc37 and Cdk4 and Coomassie stain for Hsp90. Dephosphorylation of the coprecipitated samples was confirmed by probing with α-pSer13-Cdc37. (D) The experiment was repeated in triplicate, and the amount of the components precipitated by the Talon resin (Bound) relative to the start of the incubation, was determined by densitometry of the Western blots for Cdk4 and Cdc37, or the Coomassie-stained PAGE gel for Hsp90. Error bars indicate SD of the measurements.
Figure 3
Figure 3
Dephosphorylation of Cdc37 by PP5 In Vitro (A) H-C-K complex or Sf9-expressed Cdc37 was incubated with PP5 at room temperature and the extent of dephosphorylation at specified times assessed by probing Western blots with α-pSer13-Cdc37. This experiment was repeated in the presence of a 10-fold molar excess of PP5 activator—either a seven residue peptide (SRMEEVD) corresponding to the TPR binding motif of Hsp90 or full-length human Hsp90α. (B) The interaction of PP5 with Cdc37 is mediated by Hsp90. GST-tagged PP5 was incubated with Cdc37 or phosphorylated Cdc37 in the presence and absence of Hsp90 at 4°C. A small fraction of pSer13-Cdc37 is found in complex with GST-PP5, but the majority of Cdc37 remains phosphorylated; this interaction is significantly increased in the presence of Hsp90, and coincides with an increase in PP5 activity toward Cdc37 (Unbound, lanes 6 and 8). The affinity of GST-tagged PP5 for Hsp90-bound Cdc37 is low compared with that for Hsp90-bound pSer13-Cdc37 (10% Bound, lanes 7 & 8). CBB = Coomassie Brilliant Blue. (C) The interaction of PP5 with the H-C-K complex. GST-tagged PP5 was incubated with the H-C-K complex at 4°C, and could coprecipitate all components of the complex. The interaction was stabilized, and the activity of PP5 reduced, by inclusion of AMPPNP, but not ADP.
Figure 4
Figure 4
Dephosphorylation of Cdc37p by Ppt1p in Yeast Extracts (A) Cdc37p is phosphorylated on both S14 and S17 in yeast cell extracts. Immunoprecipitated (IP) Cdc37-HA and its S14A and S17A mutants were immunoblotted with anti-Phospho-Serine antibody. (B) FLAG-tagged Ppt1p coimmunoprecipitates with the wild-type (WT), nonphosphorylatable (S14A/S17A), and phosphomimic (S14E/S17E) forms of HA-tagged Cdc37p. (C) HA-tagged wild-type Cdc37p immunoprecipitated from cells overexpressing FLAG-tagged Ppt1p is substantially dephosphorylated, as are the single S14A and S17A phosphorylation-site mutants. Overexpression of Ppt1 has no effect on the phosphorylation of HA-tagged Cdc37p in cells expressing the nonphosphorylatable S14A/S17A mutant. A FLAG-tagged Ppt1p K81E/R85E mutant, which abrogates Ppt1 interaction with Hsp90, no longer dephosphorylates HA-tagged Cdc37p.
Figure 5
Figure 5
Effects of Cdc37 Mutation and Overexpression/Deletion of Ppt1 on v-Src Expression in Yeast (A) Growth of wild-type (CDC37) cells and strains with S14A, S17A, or the doubly mutated S14E/S17E Cdc37 containing the toxic vSrc gene controlled by the GAL promoter, on glucose (glu)- or galactose (gal)-containing medium. Both nonphosphorylatable and phosphomimetic mutants of Cdc37 compromise vSrc activation. (B) The same strains analyzed for total pY and v-Src levels. Appreciable levels of v-Src were only detectable in wild-type cells. (C) Both the loss (ppt1Δ) and overexpression (PPT1+) of Ppt1p compromise v-Src expression in cells containing the toxic v-Src gene controlled by a GAL promoter. (D) The same strains analyzed for total pY and v-Src levels. As for Cdc37 phosphorylation mutants, appreciable levels of v-Src were only detectable in wild-type cells. (E) Overexpression of Ppt1p causes GA sensitivity in yeast. Cells expressing the wild-type (CDC37), nonphosphorylatable (S14A/S17A), or phosphomimic (S14E/S17E) Cdc37p were transformed with either empty vector or the PPT1 overexpression plasmid (MET25-PPT1). A 1:10 dilution series was grown (4 days, 30°C) on SD agar lacking methionine, without or with 30 μM GA. Overexpression of Ppt1 renders cells as sensitive as the nonphosphorylatable S14A, S17A mutant, of Cdc37.
Figure 6
Figure 6
PP5 Dephosphorylates Cdc37 in Tumor Cells and Compromises C-Raf Function Cell lysate of HCT116 cells stably transfected with empty vector (v) or a PP5 expression plasmid (PP5). After 3 weeks, decreased levels of total C-Raf and activated ERK (phospho-ERK1/2) were observed. The effect was amplified further after an additional week of PP5 overexpression.
Figure 7
Figure 7
Functional Cycle of Cdc37 Driven by Constitutive Phosphorylation and Targeted Dephosphorylation Schematic of coupling of CK2 phosphorylation and Hsp90-targeted dephosphorylation by PP5/Ppt1 to the role of Cdc37 in assembly and Hsp90-dependent activation of kinase clients. A C-K complex containing an inactive kinase binds an Hsp90 dimer with loss of one Cdc37 to give a stable H-C-K complex, as previously described (Vaughan et al., 2006) (a). Cdc37 is phosphorylated in both these complexes (see above). PP5/Ppt1 can bind to Hsp90 in the H-C-K complex (b) and dephosphorylate Cdc37 prior to, or concomitant with, binding of ATP and other cochaperones, such as Aha1 (e.g., [c]), generating an activated kinase and dissociating the Hsp90-based complex. Free Cdc37 is rephosphorylated by the constitutive activity of CK2 (d) to regenerate the active pSer13 form of the cochaperone, poised for reengagement in a new round of Hsp90-dependent kinase activation.
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