Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro
Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in c...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-01-01
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| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10118190/?tool=EBI |
| _version_ | 1797842317289193472 |
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| author | Daniel Koch Ay Lin Kho Atsushi Fukuzawa Alexander Alexandrovich Kutti J. Vanaanen Andrew Beavil Mark Pfuhl Martin Rees Mathias Gautel |
| author_facet | Daniel Koch Ay Lin Kho Atsushi Fukuzawa Alexander Alexandrovich Kutti J. Vanaanen Andrew Beavil Mark Pfuhl Martin Rees Mathias Gautel |
| author_sort | Daniel Koch |
| collection | DOAJ |
| description | Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation. |
| first_indexed | 2024-04-09T16:45:55Z |
| format | Article |
| id | doaj.art-833ce13322bd4ec0b24df6daefd1e60b |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-09T16:45:55Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-833ce13322bd4ec0b24df6daefd1e60b2023-04-23T05:31:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-01184Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitroDaniel KochAy Lin KhoAtsushi FukuzawaAlexander AlexandrovichKutti J. VanaanenAndrew BeavilMark PfuhlMartin ReesMathias GautelObscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10118190/?tool=EBI |
| spellingShingle | Daniel Koch Ay Lin Kho Atsushi Fukuzawa Alexander Alexandrovich Kutti J. Vanaanen Andrew Beavil Mark Pfuhl Martin Rees Mathias Gautel Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro PLoS ONE |
| title | Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro |
| title_full | Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro |
| title_fullStr | Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro |
| title_full_unstemmed | Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro |
| title_short | Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro |
| title_sort | obscurin rho gef domains are phosphorylated by mst family kinases but do not exhibit nucleotide exchange factor activity towards rho gtpases in vitro |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10118190/?tool=EBI |
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