Mahogunin-mediated α-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation

D Srivastava,O Chakrabarti

doi:10.1038/cddis.2014.1

Abstract

Mahogunin ring finger-1 (MGRN1) is a cytosolic ubiquitin ligase whose disruption or interaction with some isoforms of cytosolically exposed prion protein leads to spongiform neurodegeneration and also lack of which results in reduced embryonic viability due to mispatterning of the left–right (LR) axis during development. Here we demonstrate an interaction between the cytoskeletal protein α-tubulin and MGRN1. In cultured cell systems, loss of the ubiquitin E3 ligase activity of MGRN1 results in spindle misorientation and decreased α-tubulin polymerization, an effect also seen in primary cells. α-Tubulin was post-translationally modified by MGRN1 via noncanonical K6-linked polyubiquitination. This was significant because expression of catalytically inactive MGRN1 and/or ubiquitin mutant capable of only monoubiquitination resulted in similar mitotic spindle misorientation. The modulatory effect of MGRN1 was specific for α-tubulin and similar changes could not be detected in β- or γ-tubulin. However, catalytic inactivation of MGRN1 did not abrogate monoubiquitination of α-tubulin, thus unraveling a unique dual mode of ubiquitination by an unknown E3 ligase and MGRN1. MGRN1-mediated α-tubulin modification, and hence its stability, may highlight a key event in the LR patterning during embryogenesis.

Highlights

Recent reports indicate that Huntingtin (HTT) protein, mutations of which cause Huntington’s disease (HD), localizes to spindle poles throughout mitosis
Our analysis identifies a unique mode of ubiquitination of a-tubulin by mahogunin ring finger-1 (MGRN1) as a novel post-translation modification of this cytoskeletal protein, responsible for the formation and maintenance of the mitotic spindle fibers
This pattern of close association of MGRN1 with mitotic apparatus could be detected through the different stages of mitosis (M phase) (Supplementary Figure S1A)

Summary

Introduction

Recent reports indicate that Huntingtin (HTT) protein, mutations of which cause Huntington’s disease (HD), localizes to spindle poles throughout mitosis. Parkin–tubulin interaction in human cell lines leads to increased ubiquitination and accelerated degradation of a- and b-tubulins. Point mutants of Parkin (K161N, T240R and C431H) identified in Parkinson’s disease (PD) patients are incompetent in the E3 ligase activity toward tubulins.[9]. Recent reports indicate that BRCA1 (breast cancerassociated gene 1) protein binds to and ubiquitinates g-tubulin that is crucial for maintaining appropriate centrosome number in cells.[11,12,13,14] In addition, mutations in ubiquitin C-terminal hydrolase L1 (UCH L1), a cysteine hydrolase,[15] expressed abundantly and exclusively in brain and reproductive tissues,[16] are associated with PD and Alzheimer’s diseases (AD).[17] Studies show that besides being a deubiquitinating

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