Protein Arginine Methylation of Tubulin Beta Decreases Binding of Taxol in Neuro2a Cells

Abstract

Tubulin α and β heterodimers are the building blocks of microtubules, which are part of the cytoskeleton in eukaryotic cells. Microtubules play critical roles in both development and synaptic function in neurons and are the target of various anti‐cancer drugs. Tubulins undergo multiple post‐translational modifications which influence microtubule stability, motor protein guidance and contribute to the “microtubule code” that selectively and specifically regulates the functions of microtubules. Here we report that tubulin β is methylated on two novel arginine (R) residues. Using mass spectrometry, R62 and R282 were identified to be methylated in Neuro2a cells. The location of these methylated residues suggests a role in microtubule stability. Furthermore, R282 is involved in binding to microtubule stabilizing drugs such as taxol and epothilone derivatives and mutants of R282 are associated with anti‐cancer drug resistance. Computer modelling using protein‐docking simulations on taxol binding to tubulin β with and without methylation of R282, suggested that methylation on R282 reduces taxol binding. This prediction was supported experimentally by cell cycle analysis of Neuro2a cells using flow cytometry. Inhibition of methylation by AdOx caused an increased effect of taxol with more cells being arrested in the G2 phase. Hence our findings add a new player to the multitude of post‐translational modifications of the “microtubule code” and have potentially important implications for anti‐cancer drug design, brain development and synaptic function.