Fig 2: Mis-localisation of CNK2 also influences TNIK localisation in neurons. (A) Images of primary neurons and higher magnification image below the corresponding image expressing EGFP-CNK2 (cyan) or EGFP-CNK2ΔPH (cyan) co-stained for endogenous TNIK (magenta) and MAP2 (grey). Scale bar: upper panel: 20 µm, zoom in: 10 µm (B) Expression of EGFP-CNK2ΔPH causes a reduction of TNIK in secondary dendrites compared to EGFP or EGFP-CNK2. TNIK intensity was measured in 4–5 ROIs along secondary dendrites and normalised to MAP2 (a.u.). Data were normalised to the mean of EGFP-CNK2. Graph represents mean ± SEM of TNIK intensity (n = 11–14 neurons, N = 3 cultures): EGFP-CNK2 = 1 ± 0.04, EGFP-CNK2ΔPH = 0.87 ± 0.04, data were analysed by unpaired t-test, EGFP-CNK2 vs. EGFP-CNK2ΔPH: p = 0.0478. (C) TNIK intensity was measured in the soma of neurons expressing EGFP-CNK2ΔPH or EGFP-CNK2 and normalised to MAP2. Data were normalised to the mean of EGFP-CNK2. Graph represents mean ± SEM (a.u.) of TNIK intensity (n = 11–12 neurons, N = 3 cultures): EGFP-CNK2 = 1 ± 0.06, EGFP-CNK2ΔPH = 0.96 ± 0.06. Data were analysed by unpaired t-test, EGFP-CNK2 vs. EGFP-CNK2ΔPH: p = 0.6782.

Fig 3: TNIK localisation is regulated by CNK2. (A) Scheme of CNK2 truncation variants (for detailed description of domains see Fig. 4A legend). (B) Coimmunoprecipitation experiments of EGFP-CNK2 variants overexpressed in CHL V79 cells together with FLAG-TNIK. Proteins were immunoprecipitated with either anti-GFP (mouse) antibody or mouse IgGs as a negative control. Pull-down control and coprecipitated proteins were analysed by western blot with anti-GFP (IP) and anti-FLAG (coIP) antibodies. Input control (lysate) is shown on the left. (C) TNIK localisation is influenced by CNK2 in heterologous cells. Representative immunofluorescence experiments in CHL V79 cells overexpressing FLAG-TNIK together with EGFP as control (upper panel), EGFP-CNK2 (middle panel) or EGFP-CNK2ΔPH (lower panel). Left panels show EGFP only or EGFP-tagged CNK2 variants (cyan), middle panels show FLAG-TNIK (magenta), and the right lane shows merged channels. Scale bar: 20 µm. (D) For quantification of TNIK localisation in CHL V79 cells reflecting the experiments shown in (C), images expressing both proteins, were randomised and classified according to cytosolic or membranous TNIK localisation. Co-expression of CNK2 recruits the main fraction of FLAG-TNIK to the membrane. Upon co-expression of EGFP-tagged CNK2 variants lacking the membrane binding region, FLAG-TNIK shows essentially no membranous localisation. Data used for quantification include data for a total of 73–114 cells per condition, from three independent experiments.

Fig 4: CNK2 interacts with TNIK and the two proteins co-localise in dendritic spines. (A) Scheme of CNK1 and CNK2 domain architecture (SAM, sterile alpha motif; CRIC, conserved region in CNK; PDZ, PSD-95/DLG-1/ZO-1; DUF1170, domain of unknown function; PH, pleckstrin homology). (B) CNK2 specifically interacts with TNIK. Coimmunoprecipitation experiment with CNK1-V5, CNK2-V5 and FLAG-TNIK expressed in CHL V79 cells. Proteins were immunoprecipitated with either anti-V5 (mouse) antibody or mouse IgGs as a negative control. Proteins were detected by western blot with anti-V5 (CNK2) and anti-FLAG (TNIK) antibodies. (C) Representative image of a primary neuron (DIV 21) showing endogenous CNK2 (cyan) and endogenous TNIK (magenta) in dendritic spines. Scale bar: 10 µm (D) Coimmunoprecipitation experiment of EGFP-CNK2 with endogenous TNIK expressed in neurons. Protein was immunoprecipitated with either anti-GFP (mouse) or mouse IgGs as a negative control. Proteins were detected by western blot with anti-GFP (CNK2) and anti-TNIK antibodies. Input control (lysate) is shown on the left.