L347P PINK1 mutant that fails to bind to Hsp90/Cdc37 chaperones is rapidly degraded in a proteasome-dependent manner

Abstract

Mutation of PTEN-induced kinase 1 (PINK1), which encodes a putative mitochondrial serine/threonine kinase, leads to PARK6, an autosomal recessive form of familial Parkinson's disease. Although the precise function(s) of PINK1 protein is unknown, the recessive inheritance of this form of Parkinson's disease suggests loss of PINK1 function is closely associated with its pathogenesis. Here we report that PINK1 forms a complex with the molecular chaperones Hsp90 and Cdc37/p50 within cells, which appears to enhance its stability. When cells were treated with an Hsp90 inhibitor (geldanamycin or novobiocin), levels of PINK1 were greatly diminished, reflecting its rapid degradation via ubiquitin-proteasome pathway. Similarly, the half-life of a pathogenic PINK1 mutant (L347P) that did not interact with Hsp90 or Cdc37/p50 was only 30 min, whereas that of wild-type PINK1 was 1 h. These results strongly suggest that Hsp90 and Cdc37 are binding partners of PINK1 which regulate its stability.

Introduction

Parkinson's disease (PD) is the second most frequently occurring neurodegenerative disorder and is characterized by selective dopaminergic neural cell loss in the substantia nigra (Dauer and Przedborski, 2003). Most cases of PD are sporadic; in 5–10% of the PD patients, however, the cause is an inherited gene mutation. Moreover, the fact that the clinical characteristics of familial PD are similar to those of sporadic PD has lead to efforts to understand the pathogenic mechanisms induced by the related gene mutations. Several genes are now known to be causally associated with familial PD (Abou-Sleiman et al., 2006). Among them, mutations in the PTEN-induced putative kinase 1 gene (PINK1) have been shown to be associated with an autosomal recessive form of familial PD (Valente et al., 2004).

PINK1 was initially isolated from endometrial cancer cells overexpressing PTEN (Unoki and Nakamura, 2001), and the predicted primary sequence of PINK1 protein included an N-terminal mitochondrial-targeting signal along with a catalytic serine/threonine kinase domain. Although PINK1's mitochondrial localization and self-directed phosphorylation activity have already been characterized (Valente et al., 2004, Beilina et al., 2005, Silvestri et al., 2005, Nakajima et al., 2003), its relation to the pathogenesis of PD is poorly understood. However, evidence from several recent studies suggests that PINK1 has the ability to protect cells from stress-induced mitochondrial dysfunction and apoptosis (Valente et al., 2004, Petit et al., 2005, Deng et al., 2005, Park et al., 2006, Clark et al., 2006, Tang et al., 2006). In addition, Deng et al. (2005) recently showed that suppression of PINK1 expression reduces cell viability and significantly increases 1-methyl-4-phenylpyridinium (MPP⁺)- and rotenone-induced cytotoxicity. Consistent with those findings, PINK1-null flies exhibit male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses, including oxidative stress (Park et al., 2006, Clark et al., 2006). These data, along with the recessive nature of PINK1 mutations, suggest that this form of familial PD is associated with the loss of PINK1 function.

On the other hand, Tang et al. (2006) showed that DJ-1, another protein causatively associated with familial PD, normally interacts with and stabilizes PINK1, and DJ-1 mutations that attenuate this interaction reduce the stability of PINK1. These findings suggest that protein–protein interactions between PINK1 and one or more unknown proteins could play a key regulatory role in affecting the activity and stability of PINK1. In the present study, therefore, we endeavored to isolate PINK1-binding partners using a combination of immunoprecipitation and mass-spectrometric analysis with the aim of obtaining additional information on the pathogenic features of PINK1 mutations. Our findings suggest that the stability of PINK1 is strongly affected by its interaction with Hsp90, and that inhibition of the PINK1–Hsp90 interaction might contribute to the pathogenesis of PD.