Molecular determinants for α-tubulin methylation by SETD2

Area of Science:

• Biochemistry
• Cell Biology
• Epigenetics

Background:

• Post-translational modifications of tubulin regulate microtubule functions.
• SETD2 methylates histone proteins and was recently found to methylate tubulin, impacting mitosis.
• The biochemical mechanism of tubulin methylation by SETD2 remains poorly understood.

Purpose of the Study:

• To elucidate the catalytic mechanism of tubulin methylation by SETD2.
• To investigate the substrate recognition mechanisms of SETD2 for both histone and tubulin.
• To understand the distinct roles of SETD2 domains in dual substrate methylation.

Main Methods:

• Utilized a truncated human wild-type SETD2 (tSETD2) enzyme.
• Employed recombinant single-isotype tubulin and polymerized microtubules.
• Introduced pathogenic mutations into tSETD2 catalytic and SRI domains.
• Investigated substrate binding affinities and methylation activity.

Main Results:

• Recombinant tSETD2 exhibited higher activity towards tubulin dimers than polymerized microtubules.
• Tubulin methylation by SETD2 was specifically localized to lysine 40 of α-tubulin.
• Mutations in SETD2's catalytic domain impaired methylation but not binding, while SRI domain mutations affected both.
• SETD2's SRI domain showed differential binding to tubulin versus RNA polymerase II, suggesting distinct substrate recognition pathways.
• The C-terminal tail of α-tubulin is essential for SETD2 substrate recognition.

Conclusions:

• This study provides a mechanistic framework for SETD2's dual methyltransferase activity on histones and tubulin.
• Distinct mechanisms govern SETD2-mediated methylation of tubulin and histones.
• Understanding these mechanisms is key to comprehending microtubule-based cellular functions and mitotic integrity.