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Updated: Jan 15, 2026
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Regulation of death-associated protein kinase. Stabilization by HSP90 heterocomplexes
Liguo Zhang1, Kenneth P Nephew, Patricia J Gallagher
1Department of Cellular and Integrated Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Abstract:
Death-associated protein kinase (DAPK) has been found associated with HSP90, and inhibition of HSP90 with 17-alkylamino-17-demethoxygeldanamycin reduced expression of DAPK. These results were extended to determine whether the degradation of DAPK in the absence of HSP90 activity is dependent on the ubiquitin-proteasome pathway. Our results show that treatment of cells with geldanamycin (GA) leads to degradation of DAPK, and this degradation is attenuated by the proteasome inhibitor, lactacystin. GA-induced DAPK degradation is also dependent on phosphorylation of DAPK at Ser(308), and the cellular levels of phospho(Ser(308))-DAPK dramatically increase in response to GA treatment. Expression of two distinct ubiquitin E3 ligases, carboxyl terminus of HSC70-interacting protein (CHIP) or DIP1/Mib1, enhanced DAPK degradation, and conversely, short interfering RNA depletion of either CHIP or DIP1/Mib1 attenuated DAPK degradation. In vitro ubiquitination assays confirmed that DAPK is targeted for ubiquitination by both CHIP and DIP. Consistent with these results, DAPK is found in two distinct immune complexes, one containing HSP90 and CHIP and a second complex containing only DIP1/Mib. Collectively, these results indicate that strict modulation of DAPK activities is critical for regulation of apoptosis and cellular homeostasis.
Insights
Heat shock protein 90 (HSP90) inhibition triggers the ubiquitin-proteasome pathway to degrade death-associated protein kinase (DAPK). This degradation is mediated by E3 ligases CHIP and DIP1/Mib1, highlighting DAPK regulation in cellular homeostasis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Death-associated protein kinase (DAPK) plays a role in apoptosis and cellular homeostasis.
- HSP90 is known to interact with and influence the stability of various proteins.
Purpose of the Study:
- To investigate the mechanism of DAPK degradation following HSP90 inhibition.
- To determine the role of the ubiquitin-proteasome pathway in DAPK regulation.
Main Methods:
- Cell treatment with HSP90 inhibitor (geldanamycin) and proteasome inhibitor (lactacystin).
- Analysis of DAPK phosphorylation at Ser(308).
- Overexpression and siRNA depletion of E3 ligases (CHIP, DIP1/Mib1).
- In vitro ubiquitination assays and co-immunoprecipitation.
Main Results:
- HSP90 inhibition by geldanamycin leads to DAPK degradation, which is blocked by lactacystin.
- DAPK degradation is dependent on phosphorylation at Ser(308), with increased phospho-DAPK levels upon geldanamycin treatment.
- E3 ligases CHIP and DIP1/Mib1 enhance DAPK degradation, while their depletion attenuates it.
- DAPK is ubiquitinated by both CHIP and DIP1/Mib1 and forms complexes with HSP90/CHIP and DIP1/Mib1.
Conclusions:
- DAPK degradation is mediated by the ubiquitin-proteasome system upon HSP90 inhibition.
- Phosphorylation at Ser(308) and E3 ligases CHIP and DIP1/Mib1 are critical for this degradation process.
- Regulation of DAPK activity through this pathway is essential for maintaining cellular homeostasis and apoptosis control.
