Fig. 3
TLK2 undergoes both cis- and trans-phosphorylation in the dimer. a Hierarchical clustering analysis of the phosphorylation sites of the ΔN-TLK2 constructs expressed in E. coli displayed in a heat map. Log-transformed and row-normalized intensities of phosphosites are shown in triplicates for the heterodimer kinase-dead subunit (HeteroKD), the heterodimer active subunit (Hetero), the active homodimer (ΔN-TLK2) and the kinase-dead homodimer (ΔN-TLK2-KD). b Volcano plot validation showing the regulation and significance of phosphosites between the ΔN-TLK2 homodimer and the kinase-dead ΔN-TLK2-KD homodimer. Phosphosites are labelled in black (significant), light orange (highly significant) and red (most highly significant). c All detected phosphorylation sites mapped on the ΔN-TLK2 domain scheme. The drawing does not imply a parallel arrangement of the dimer. Sites cannot be assigned to individual molecules of the dimer. Boxed phosphosites have been detected in both HEK293 and E. coli-expressed ΔN-TLK2, while unboxed sites were only found in the E. coli-expressed ΔN-TLK2. Font colour represents significance as shown in Fig. 3b for the E. coli-expressed ΔN-TLK2 (black, light orange and red). The unboxed green sites represent phosphosites observed exclusively in the ΔN-TLK2 expressed in HEK293 cells. d Schematic representation depicting the unique phosphorylation sites observed in the heterodimer ΔN-TLK2 active subunit and in the ΔN-TLK2-KD kinase-dead subunit. The phosphorylation sites in T208, T213, S218, S226, S289, T300, S307, T357, T380, S761 and T762 were observed in both subunits of the heterodimer (see Supplementary Data 1 and Fig. 6a–d for volcano plots displaying distributions of peptides when comparing the different constructs)
