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.2017 Oct 31;21(5):1386-1398.
doi: 10.1016/j.celrep.2017.10.042.

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System

Affiliations

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System

Stephen T Hallett et al. Cell Rep..

Abstract

Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions.

Keywords: CDK; CIP/KIP; Cdc37; Hsp90; INK; chaperone; cyclin D; kinase; palbociclib; ribociclib.

Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract
Figure 1
Figure 1
CDK4 and CDK6 Bind to Cdc37 (A) Homogeneous time-resolved fluorescence (HTRF) assay format. (B) Cdc37 binding to CDK4 (blue line) and to CDK6 (red line) measured by HTRF. The concentration of CDK4 and CDK6 used in these assays was 10 nM. From SPR titrations between GSTCDK4 or GSTCDK6 and Cdc37, it can be calculated that 34% and 20%, respectively, of the CDK4 and CDK6 are active (Figures S1C and S1D). (C and D) ATP-competitive inhibitors can displace Cdc37 from a CDK4-Cdc37 (C) or CDK6-Cdc37 (D) complex. CDK4 and CDK6 concentrations were 8 nM and 6 nM, respectively. HTRF measurements were carried out in duplicate and repeated on 3 separate days. The error bars indicate SD. See also Figure S1.
Figure 2
Figure 2
Cyclin and Cdc37 Binding to CDKs Are Mutually Exclusive (A and B) Kcyclin (red) and Vcyclin (blue) readily displace Cdc37 from CDK6 (B) but not from CDK4 (A). The concentrations of CDK4- and CDK6-containing cyclin D complexes used in the assays were 8 nM (CDK4) and 6 nM (CDK6), respectively. HTRF measurements were carried out in duplicate and repeated on 3 separate days. The error bars indicate SD. (C and D) FLAG-tagged Cdc37 was co-incubated with CDK4-cyclin D1/D3 (C) or CDK6-cyclin D1/D3 (D) and then analyzed by SDS-PAGE and subsequent InstantBlue staining. The upper band present in the CDK6-cyclin samples is CDK6, whereas the lower band is the cyclin D. The upper band of the CDK4-cyclin D1 complex is cyclin D1, whereas the lower band is CDK4. CDK4 and cyclin D3 have very similar masses and, when analyzed together, are not resolved. Inputs are marked by the black bars above the right hand side lanes. CDK4, but not cyclin D1 or D3, is detected bound to the FLAG-tagged Cdc37 beads (C), indicating that Cdc37 has displaced CDK4 from either cyclin partner. In contrast, CDK6 is not detectable bound to Cdc37 when bound to cyclin D3 but is displaced from cyclin D1 following incubation with Cdc37 (D). (E and F) Kcyclin binds to CDK4 (E) and CDK6 (F) and is able to displace CDK6 (F) but not CDK4 (E) from CDK-Cdc37 and CDK-Cdc37-Hsp90β complexes assembled in insect cells. The uncropped gels that include the control lanes to confirm that CDK4, CDK6, Cdc37, and Hsp90 do not stick non-specifically to Avi-tagged beads are included as Figures S2G and S2H. Inputs are marked by the black bars above the right hand side lanes. Samples were analyzed by SDS-PAGE and subsequent InstantBlue staining. See also Figure S2.
Figure 3
Figure 3
CKI-Mediated Displacement of Cdc37 from CDK4-Cdc37 and CDK6-Cdc37 Complexes (A and B) Displacement of Cdc37 from CDK4 (A) or CDK6 (B) by members of the INK family. (C and D) p16INK4a is able to displace CDK4 (C) and CDK6 (D) from CDK-Cdc37 and CDK-Cdc37-Hsp90β complexes assembled in insect cells. The uncropped gels that include the control lanes to confirm that CDK4, CDK6, Cdc37, and Hsp90 do not stick non-specifically to Avi-tagged beads are included as Figure S4. (E and F) Mutant p16INK4a varies in its ability to displace Cdc37 from a CDK4-Cdc37 complex (E) or a CDK6-Cdc37 complex (F). The concentrations of CDK4 and CDK6 were 8 nM and 6 nM, respectively. The HTRF measurements presented in (A) and (B) were carried out in duplicate and repeated on 3 separate days. The measurements presented in (C) and (D) were carried out in duplicate and independently repeated twice. The error bars indicate SD. See also Figures S3 and S4.
Figure 4
Figure 4
p21CIP1 and p27KIP1 Cooperate with Cyclin D to Prevent Cdc37 Association with CDK4 and CDK6 (A and B) p27KIP1FL (p27FL), p21CIP1S/M (p21S, p21M), and p27KIP1S/M (p27S, p27M) can displace CDK4 (A) or CDK6 (B) from Cdc37. (C and D) Cdc37 binds to CDK4 (C) or CDK6 (D) (blue curves) and can effectively displace cyclin D1 (red curves). Upon addition of excess Cdc37, the ternary CDK-cyclin D1-p27KIP1M complexes do not redistribute to form CDK-Cdc37 (green curves). (E) Cdc37 binds to CDK4 (blue) and can effectively displace cyclin D3 bound to CDK4 (red). Upon addition of excess Cdc37, ternary complexes of CDK4-cyclin D3-p27KIP1M or p21CIP1M do not redistribute to form CDK4-Cdc37 (green and magenta curves, respectively). (F) In contrast, CDK6 binds to Cdc37 (blue curve) but cannot be displaced from cyclin D3 by Cdc37 (red curve). All HTRF measurements were carried out in duplicate and repeated. The error bars indicate SD.
Figure 5
Figure 5
Exchange of CDKs into and out of Complexes Containing Cdc37 The experiments reconstitute the redistribution of CDKs 4 and 6 (blue) between complexes that contain Cdc37 (coral) and complexes that contain other binding partners drawn from ATP-competitive inhibitors (red), cyclins (green), CIP/KIP family CKIs (yellow), and INK family CKIs (purple). Boxed half arrows indicate the half reactions of these equilibria that have been reconstituted in the present study. Smaller arrows in the “reverse” direction (i.e., toward complexes that contain Cdc37) indicate reactions where the competitor appears to bind to CDKs substantially more tightly than does Cdc37. Partitioning between the different partnered states is presumed to proceed via unbound states of the kinase, which exist in an equilibrium between less folded “open” states (which preferentially bind to the chaperone system) and more folded “closed” states (which preferentially bind to the other partners).
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