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 3 The effect of never in mitosis gene A-related kinase 5 on the activity of different truncations of the tubulin tyrosine ligase-like 4. A: Schematic representation of the tubulin tyrosine ligase-like (TTLL) 4 constructs used. The sequences expressed for each truncated version used in this work are depicted as black bars underneath the pictograms of the full-length TTLL proteins. Domains unnecessary or required for autonomous activities are shown. Green area corresponds to tubulin tyrosine ligase domain (599-942 a.a.), the purple area is extended tubulin tyrosine ligase domain, the blue area is the ATP binding site and the orange construction is the TTLL4 sequence identified by yeast two-hybrid screening. See text for more details; and B-D Analysis by Western blot of truncations of TTLL4 and TTLL5 promoting polyglutamylation in (b) control, (c) cells stably expressing never in mitosis gene A-related kinase 5-kinase-dead or (d) cells silenced for expression of never in mitosis gene A-related kinase 5. NEK5: Never in mitosis gene A-related kinase 5; KD: Kinase-dead; shRNA: Short hairpin ribonucleic acid; EYFP: Enhanced yellow fluorescent protein; TTLL: Tubulin tyrosine ligase-like.