Parkin Binds to α/β Tubulin and Increases their Ubiquitination and Degradation

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Abstract
In addition to inhibiting the mitochondrial respiratory chain, toxins known to cause Parkinson9s disease (PD), such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, also strongly depolymerize microtubules and increase tubulin degradation. Microtubules are polymers of tubulin α/β heterodimers, whose correct folding requires coordinated actions of cellular chaperonins and cofactors. Misfolded tubulin monomers are highly toxic and quickly degraded through a hitherto unknown mechanism. Here we report that parkin, a protein–ubiquitin E3 ligase linked to PD, was tightly bound to microtubules in taxol-mediated microtubule coassembly assays. In lysates from the rat brain or transfected human embryonic kidney (HEK) 293 cells, α-tubulin and β-tubulin were strongly coimmunoprecipitated with parkin at 4°C in the presence of colchicine, a condition in which tubulin exits as α/β heterodimers. At the subcellular level, parkin exhibited punctate immunostaining along microtubules in rat brain sections, cultured primary neurons, glial cells, and cell lines. This pattern of subcellular localization was abolished in cells treated with the microtubule-depolymerizing drug colchicine. The binding between parkin and tubulin apparently led to increased ubiquitination and accelerated degradation of α- and β-tubulins in HEK293 cells. Similarly ubiquitinated tubulins were also observed in rat brain lysates. Furthermore, parkin mutants found in PD patients did not ubiquitinate or degrade either tubulin. Taken together, our results show that parkin is a novel tubulin-binding protein, as well as a microtubule-associated protein. Its ability to enhance the ubiquitination and degradation of misfolded tubulins may play a significant role in protecting neurons from toxins that cause PD.