title: Oncogenic RET kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans.
abstract: To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation.
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK. Electronic address: ivan.plaza-menacho@cancer.org.uk.
authors.forename: Iván
authors.initials: I
authors.lastname: Plaza-Menacho
authors.affiliation: Protein Analysis and Proteomics, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Karin
authors.initials: K
authors.lastname: Barnouin
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Kerry
authors.initials: K
authors.lastname: Goodman
authors.affiliation: Protein Structure Function Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Rubén J
authors.initials: RJ
authors.lastname: Martínez-Torres
authors.affiliation: Protein Production Facility, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Annabel
authors.initials: A
authors.lastname: Borg
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Judith
authors.initials: J
authors.lastname: Murray-Rust
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Stephane
authors.initials: S
authors.lastname: Mouilleron
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK.
authors.forename: Phillip
authors.initials: P
authors.lastname: Knowles
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, WC2A 3LY London, UK; Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, WC1E 7HX London, UK. Electronic address: neil.mcdonald@cancer.org.uk.
authors.forename: Neil Q
authors.initials: NQ
authors.lastname: McDonald
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007313:Insecta; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051096:Proto-Oncogene Proteins c-ret / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human