Parkin Stabilizes Microtubules through Strong Binding Mediated by Three Independent Domains

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 ␣/␤ 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 microtubulebinding 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.

Parkin regulates paclitaxel sensitivity in breast cancer via a microtubule‐dependent mechanism

“…To test this possibility, MCF7 cells transfected with GFP or GFP-Parkin were treated with paclitaxel and examined by immunofluorescence microscopy. Consistent with previous studies [14,23], overexpression of GFP or GFP-Parkin or treatment with sub-nanomolar concentrations of paclitaxel did not obviously affect the morphology of cellular microtubules (Figure 2A). Strikingly, in cells overexpressing GFP-Parkin, upon paclitaxel treatment 55% of cells exhibited microtubule bundles (Figure 2A, B), indicating that Parkin increases the ability of paclitaxel to promote microtubule assembly.…”

“…In addition, we find that the effect of Parkin on paclitaxel sensitivity is abolished by deletion of the microtubulebinding domain. Given that Parkin binds to microtubules with high affinity [14], these findings suggest that the activity of Parkin towards paclitaxel sensitivity is mediated specifically by a microtubule-dependent mechanism.…”

“…Parkin partly co-localizes with microtubules in cells and is present in purified microtubule preparations. The association of Parkin with microtubules appears to be strong and endures high concentrations of salt and urea [14]. In addition, the association is not affected by mutations that abolish the ubiquitin ligase activity of Parkin [13,14].…”

“…The association of Parkin with microtubules appears to be strong and endures high concentrations of salt and urea [14]. In addition, the association is not affected by mutations that abolish the ubiquitin ligase activity of Parkin [13,14]. It has been suggested that microtubules may serve to anchor Parkin in the cytoplasm and thereby regulate its enzymatic activity [15].…”

“…In addition to its function as a ubiquitin ligase, Parkin has recently been identified as a molecule capable of interacting with microtubules [13,14]. Parkin partly co-localizes with microtubules in cells and is present in purified microtubule preparations.…”