Fig 1.
Identification of Warburg-Cinotti Syndrome associated DDR2 mutations.
A. Schematic of the DDR2 cytosolic region (amino acids 400 to 855) with amino acid boundaries of the JM4 region and the kinase indicated. TM, transmembrane domain. B. Left, Crystal structure of DDR2 kinase domain. Figure generated with PyMOL using PDB code 7AYM [36]. The A-loop is shown in green. The αC helix is shown in cyan and labelled with ‘αC’. Residues that are mutated in Warburg-Cinotti syndrome, are labelled as ‘Leu610’ and ‘Tyr740’, with Leu610 in blue located before the αC helix and Tyr740 in brown on the A-loop. Right, Close-up view of the residues that are mutated in Warburg-Cinotti Syndrome. Mutagenesis prediction was performed using PyMOL. Selected side chains are presented in atomic detail. Upper left, β3 strand residue Lys608 (in yellow) forms a salt bridge with the αC helix residue Glu625 (in yellow). Leu610 is presented in blue with the side chain positioned towards the αC helix; Upper right, the predicted position of Pro610 is shown in red. Lower left, WT Tyr740 (in brown) forms hydrogen bonds with Asp710 and Arg714 (both in yellow). The A-loop is presented in green. Lower right, in the Y740C variant, the hydrogen bonds with Asp710 and Arg714 are no longer present due to the substitution with Cys740. NB: this panel is rotated relative to the left-hand panel in order to visualise better the relevant interactions.
Fig 2.
Full-length DDR2-Y740C and DDR2-L610P are constitutively active in the absence of collagen stimulation.
A. Left, Schematic of the DDR2 architecture, showing the extracellular DS domain followed by a DS-like domain, an extracellular juxtamembrane (EJM) region and a TM domain. The intracellular part is composed of a flexible intracellular juxtamembrane (IJM) region and a catalytic TKD. The kinase A-loop is represented in green. A. Right, Expression constructs encoding full-length DDR2-WT, DDR2-Y740C, or DDR2-L610P were transfected into HEK293 cells. Cells were cultured for 24 hours, and then serum starved for at least 16 hours. Cells were then stimulated with 10 μg/mL soluble collagen I at 37°C for 90 min. Cell lysates were analysed by SDS-PAGE and Western blotting with anti-pY antibody 4G10 (binds to phospho-tyrosines independent of context) or the DDR-specific anti-pY JM4 #1 antibody (binds to pY538 in DDR2). Total DDR2 levels were then detected using an anti-DDR2 antibody. The positions of molecular weight markers (in kDa) are shown on the left. Experiments were repeated three times with similar results. B. Quantification of the anti-pY (4G10) antibody signals normalised to respective anti-DDR2 signals. The anti-pY signal is expressed as a percentage of the sum of all bands on a blot, with mean and standard error of the mean shown (n = 3). Statistically significant differences (P < 0.05) between signals are indicated by asterisks (**P = 0.0060; ***P = 0.0007; ****P < 0.0001; ns, non-significant). Two-way ANOVA with Tukey’s multiple comparisons test.
Fig 3.
Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay).
A. Schematic diagrams showing the location of key JM4 and A-loop tyrosine residues in DDR1-K-WT and DDR2-K-WT constructs. Anti-pY JM4 #1 binds to pY569 in DDR1 and to pY521 in DDR2. Anti-pY JM4 #2 binds to pY586 in DDR1 and to pY538 in DDR2. Anti-pY A-loop antibody was raised against a phosphopeptide containing the human DDR2 Y740 site. This region is highly conserved between DDR1 and DDR2 and contains Y792, Y796, Y797 in DDR1 and Y734, Y740, Y741 in DDR2. B. The in vitro autophosphorylation assay was performed by incubating 1 μM protein constructs with 1 mM ATP in kinase buffer I at 20°C over a 60 min time course. The reactions were stopped at indicated time points by boiling with sample buffer. Samples were then analysed by SDS-PAGE and Western blotting with the JM4-specific and A-loop-specific anti-pY antibodies, as indicated. Total DDR levels were detected using anti-DDR1 or anti-DDR2 antibodies. The positions of molecular weight markers (in kDa) are shown on the left. Experiments were repeated three times with similar results.
Fig 4.
Stepwise kinase activation of DDR1 and DDR2 (100 μM protein autophosphorylation assay).
The in vitro autophosphorylation assay was performed by incubating 100 μM DDR1-K-WT or DDR2-K-WT constructs with 20 mM ATP in kinase buffer II at 20°C over a 180 min time course. The reactions were stopped at indicated time points by adding 80 mM EDTA (for native-PAGE) or boiling with sample buffer (for SDS-PAGE). Samples were either separated by native-PAGE with Coomassie staining (upper image) or analysed by SDS-PAGE and Western blotting with the JM4-specific and A-loop-specific anti-pY antibodies (lower image). Differently phosphorylated DDR forms are indicated with grey arrows. Total DDR levels were detected using anti-DDR1 and anti-DDR2 antibodies. The positions of molecular weight markers (in kDa) are shown on the left. The experiment was performed once.
Fig 5.
DDR2-Y740C kinase is more active than DDR2-WT kinase.
A. Schematic structure of the recombinant DDR2 constructs. JM4 tyrosines (Y521, Y538) and A-loop tyrosines (replaced by C740 in DDR2-K-Y740C) are labelled. The soluble DDR2-K-WT and DDR2-K-Y740C proteins were incubated at 0.1 µM in the presence of kinase buffer I and 1 mM ATP for the indicated time course (0-15 min) at 20°C. The samples were then boiled in sample buffer and analysed by SDS-PAGE and Western blotting. The positions of molecular weight markers are shown in kDa on the left side of each blot. Experiments were repeated three times with similar results. B. Quantification of the anti-pY JM4 #1 and anti-pY A-loop, signals normalised to respective anti-DDR2 signals. The anti-pY signal is expressed as a percentage of the sum of all bands on a blot, with mean and standard error of the mean shown (n = 3). Statistically significant differences (P < 0.05) between the DDR2-K-WT and DDR2-K-Y740C signals are indicated by asterisks (left graph, *P = 0.0389, t = 0.25 min; *P = 0.0270, t = 0.5 min; *P = 0.0400, t = 1 min. Right graph, **P = 0.0067, t = 0.25 min; *P = 0.0170, t = 0.5 min; **P = 0.0099, t = 5 min). Two-way ANOVA with Šídák’s multiple comparisons test. C. The soluble DDR1 and DDR2 kinase constructs, DDR1-K-WT, DDR2-K-WT, DDR1-K-WT-FP, and DDR2-K-Y740C were analysed by differential scanning fluorimetry. Proteins at 10 μM concentration were heated with SYPRO Orange from 24 to 94°C with the fluorescence detected. The average fluorescence from three independent experiments is shown. The data were analysed by Protein Thermal Shift software (ThermoFisher) using Boltzmann equation to calculate the denaturation midpoint. GraphPad Prism 9 was used for figure generation. *The DDR1-K-WT-FP protein is the purified fully phosphorylated protein as generated previously [29].
Table 1.
Apparent Km, vmax and kcat, values for Axltide peptide and ATP.
Fig 6.
A-loop cysteine mutation increases the enzymatic activity of DDR2 kinase.
A. DDR1 and DDR2 FP forms were obtained by incubating 100 μM DDR1-K-WT or DDR2-K-WT protein constructs with 20 mM ATP in kinase buffer II at 23°C for 4 hours. The reactions were stopped by adding 80 mM EDTA. Sample aliquots were either separated by native-PAGE with Coomassie staining (upper image) or analysed by SDS-PAGE and Western blotting with the JM4-specific and A-loop-specific anti-pY antibodies (lower image). DDR FP forms are indicated with grey arrows. Total DDR levels were detected using anti-DDR1 and anti-DDR2 antibodies. The positions of molecular weight markers (in kDa) are shown on the left. B. In vitro kinase activity of soluble DDR1 and DDR2 constructs was assessed using the ADP-GloTM and ADP-GloTM Max assays. The reactions were performed at 22°C and stopped at 10, 20, and 40 minutes to determine initial velocities. Top, assay performed with a range of Axltide peptide concentrations (0-520 μM) in the presence of a fixed ATP concentration (1 mM). Bottom, assay performed over a range of ATP concentrations (0-1667 μM) in the presence of a fixed Axltide peptide concentration (520 µM). Data were then fitted with the Michaelis-Menten equation. Mean and standard error of the mean shown (n = 3). SA, specific activity.