The ubiquitin E3 ligase CHIP promotes proteasomal degradation of the serine/threonine protein kinase PINK1 during staurosporine-induced cell death

Lang Yoo,Kwang Chul Chung

doi:10.1074/jbc.m117.803890

Abstract

Mutations in the gene for the serine/threonine protein kinase PTEN-induced putative kinase 1 (PINK1) are the second most frequent cause of autosomal recessive Parkinson's disease (PD). Via its kinase activity, PINK1 regulates neuronal cell survival and mitochondrial quality control. Numerous reports have revealed that PINK1 has diverse and physiologically significant functions, and therefore its activity should be tightly regulated. However, the molecular mechanisms regulating PINK1 stability and the modulator(s) involved have not been elucidated. In this study, we demonstrate that the ubiquitin E3 ligase carboxyl terminus of Hsp70-interacting protein (CHIP) promotes PINK1 ubiquitination and decreases its steady-state levels. Moreover, PINK1 levels were strongly reduced in HEK293 and SH-SY5Y cells exposed to the apoptosis-inducer staurosporine. Of note, we found that this reduction resulted from CHIP-mediated PINK1 ubiquitination. Accordingly, siRNA-mediated CHIP knockdown reduced susceptibility to staurosporine-induced cell death. Taken together, these findings suggest that CHIP plays a role in negative regulation of PINK1 stability and may suppress PINK1's cytoprotective effect during staurosporine-induced mammalian cell death. We propose that this PINK1 regulatory pathway might contribute to Parkinson's disease pathogenesis.

Highlights

Parkinson’s disease (PD)2 is a progressive neurodegenerative disorder (NDD) characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta [1]
Based on the previous finding that the upstream regulators of PINK1 stability are in some way associated with the chaperone machinery and proteasomal degradation [11], we examined biochemical and functional interactions of PINK1 with chaperonedependent ubiquitin E3 ligase carboxyl terminus of Hsp70-interacting protein (CHIP)
To first determine whether PINK1 and CHIP physically interact in mammalian cells, we performed co-immunoprecipitation analysis of lysates of cells transfected with plasmid encoding Myc-tagged PINK1 alone or together with plasmid encoding Xpress-tagged CHIP

Summary

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Because PINK1 functions in a prosurvival pathway, its activity must be tightly regulated upstream, and a complete understanding of this regulatory mechanism is critical to understanding the pathogenesis of PD. Because PINK1 processing, intracellular location, and activity are differentially regulated by interaction with Hsp, BAG2, or BAG5, respectively, it is highly likely that Hsp70/90-dependent CHIP affects the stability of PINK1 (10 –12). E, representative confocal images of immunostaining of a SH-SY5Y cell expressing both Myc-PINK1 (red) and HA-CHIP (blue). A decrease in the steady-state level of PINK1 as a result of CHIP-mediated ubiquitination was observed during staurosporine (STS)-induced cell death. These data imply that the biochemical interaction between CHIP and PINK1 and their functional linkage may play a role in STSinduced mammalian cell death and, possibly, in the pathogenesis of PD

Results

Discussion

Experimental procedures