DDR2-Y740C kinase is more active than DDR2-WT kinase.

<p><b>A.</b> 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...

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Opis bibliograficzny
1. autor:	Ziteng Hao (22647882) (author)
Kolejni autorzy:	Birgit Leitinger (111622) (author)
Wydane:	2025
Hasła przedmiotowe:	Biophysics Biochemistry Cell Biology Molecular Biology Biotechnology Immunology Infectious Diseases Virology Biological Sciences not elsewhere classified Chemical Sciences not elsewhere classified substrate phosphorylation rates mechanistic studies addressing key catalytic residues increased catalytic rates autosomal dominant manner atp binding affinity independent constitutive autophosphorylation missense variant bypasses y740c kinase constructs wt ddr2 kinase y740c kinase substituted variant missense mutations enhanced autophosphorylation ddr2 kinase ddr1 kinase y740c displayed wt ddr2 soluble wt xlink "> unphosphorylated ddr2 structural explanation skin fusion previously hypothesised mammalian cells length proteins keloid plaques function mechanism enzyme kinetics corneal vascularisation cinotti variants cinotti syndrome cause disease autoinhibitory constraints
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Opis
Streszczenie:	<p><b>A.</b> 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>B.</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. <b>C.</b> 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0336895#pone.0336895.ref029" target="_blank">29</a>].</p>

DDR2-Y740C kinase is more active than DDR2-WT kinase.

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