Parkin Stabilizes Microtubules through Strong Binding Mediated by Three Independent Domains

Fang Yang,Qian Jiang,Jinghui Zhao,Yong Ren,Mark D Sutton,Jian Feng

doi:10.1074/jbc.m500843200

Fang Yang, Qian Jiang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m500843200

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Abstract

Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Our previous studies have demonstrated that parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. Here we show that the strong binding between parkin and tubulin, as well as that between parkin and microtubules, was mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuated colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules were not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates.

Highlights

Parkinson’s disease (PD)1 is the most prevalent neurodegenerative movement disorder, affecting about 1–2% of people over 60 years of age [1]
Anti-FLAG blot of ␣-tubulin immunoprecipitates and 1% of the input lysates showed that the Linker (L), RING1 (R1), or RING2 (R2) domain was strongly co-immunoprecipitated with tubulin
This is consistent with our previous result that the binding between parkin and tubulin ␣/␤ heterodimer is not affected by colchicine [11] and suggests that each of the three domains (Linker, RING1, and RING2) binds to tubulin heterodimer at 4 °C

Summary

Introduction

Parkinson’s disease (PD) is the most prevalent neurodegenerative movement disorder, affecting about 1–2% of people over 60 years of age [1]. Apart from its E3 ligase activity on tubulin, the strong binding between parkin and microtubules may anchor this E3 ligase on microtubules to serve its functions, as misfolded proteins are known to be transported along microtubules to the aggresome [23]. Expression of the domains that bound to tubulin and microtubules significantly reduced colchicine-induced microtubule depolymerization, suggesting that parkin may stabilize the microtubule network. We found that PD-linked mutations of parkin (K161N, T240R, and C431F) did not significantly impair the binding of parkin with tubulin heterodimers or microtubules, nor did they affect the ability of parkin to stabilize microtubules against colchicine-induced depolymerization. The strong binding of parkin to microtubules may stabilize the microtubule network and anchor this E3 ligase to effectively ubiquitinate misfolded proteins being transported along microtubules

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