Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay).
<p><b>A.</b> 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 ra...
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2025
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| _version_ | 1851480994608054272 |
|---|---|
| author | Ziteng Hao (22647882) |
| author2 | Birgit Leitinger (111622) |
| author2_role | author |
| author_facet | Ziteng Hao (22647882) Birgit Leitinger (111622) |
| author_role | author |
| dc.creator.none.fl_str_mv | Ziteng Hao (22647882) Birgit Leitinger (111622) |
| dc.date.none.fl_str_mv | 2025-11-19T18:26:29Z |
| dc.identifier.none.fl_str_mv | 10.1371/journal.pone.0336895.g003 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/figure/Stepwise_kinase_activation_of_DDR1_and_DDR2_1_M_protein_autophosphorylation_assay_/30658476 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | 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 |
| dc.title.none.fl_str_mv | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| dc.type.none.fl_str_mv | Image Figure info:eu-repo/semantics/publishedVersion image |
| description | <p><b>A.</b> 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>B.</b> The <i>in vitro</i> 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.</p> |
| eu_rights_str_mv | openAccess |
| id | Manara_08750c4b7a3e2b1d2f9fcacb8b94e61f |
| identifier_str_mv | 10.1371/journal.pone.0336895.g003 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/30658476 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay).Ziteng Hao (22647882)Birgit Leitinger (111622)BiophysicsBiochemistryCell BiologyMolecular BiologyBiotechnologyImmunologyInfectious DiseasesVirologyBiological Sciences not elsewhere classifiedChemical Sciences not elsewhere classifiedsubstrate phosphorylation ratesmechanistic studies addressingkey catalytic residuesincreased catalytic ratesautosomal dominant manneratp binding affinityindependent constitutive autophosphorylationmissense variant bypassesy740c kinase constructswt ddr2 kinasey740c kinasesubstituted variantmissense mutationsenhanced autophosphorylationddr2 kinaseddr1 kinasey740c displayedwt ddr2soluble wtxlink ">unphosphorylated ddr2structural explanationskin fusionpreviously hypothesisedmammalian cellslength proteinskeloid plaquesfunction mechanismenzyme kineticscorneal vascularisationcinotti variantscinotti syndromecause diseaseautoinhibitory constraints<p><b>A.</b> 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>B.</b> The <i>in vitro</i> 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.</p>2025-11-19T18:26:29ZImageFigureinfo:eu-repo/semantics/publishedVersionimage10.1371/journal.pone.0336895.g003https://figshare.com/articles/figure/Stepwise_kinase_activation_of_DDR1_and_DDR2_1_M_protein_autophosphorylation_assay_/30658476CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/306584762025-11-19T18:26:29Z |
| spellingShingle | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). Ziteng Hao (22647882) 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 |
| status_str | publishedVersion |
| title | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| title_full | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| title_fullStr | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| title_full_unstemmed | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| title_short | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| title_sort | Stepwise kinase activation of DDR1 and DDR2 (1 μM protein autophosphorylation assay). |
| topic | 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 |