Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A -related kinase 5

Figure 5 Tyrosine 815 is important for tubulin tyrosine ligase-like 4 activity. Based on the residues identified by mass spectrometry, phosphomimetic or inactivating point mutants (Ser/Thr/Tyr to Alanine, Phenylalanine) were generated. A: Schematic representation of the tubulin tyrosine ligase-like 4 and sites of target amino acids for point mutations. The green region corresponds to the tubulin tyrosine ligase domain; and B: HEK293T proteins of cells transfected with enhanced yellow fluorescent protein-tubulin tyrosine ligase-like 4 containing different mutations were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes and stained with GT335, anti-green fluorescent proteins and anti-tubulin antibodies. GFP: Green fluorescent proteins; shRNA: Short hairpin ribonucleic acid; EYFP: Enhanced yellow fluorescent protein; TTLL: Tubulin tyrosine ligase-like; TTL: Tubulin tyrosine ligase.