title: Multi-phosphorylation of the intrinsically disordered unique domain of c-Src studied by in-cell and real-time NMR spectroscopy.
abstract: Intrinsically disordered regions (IDRs) are preferred sites for post-translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase-phosphatase networks. Here we used real-time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the "unique domain" of c-Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin-dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP-dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK-dependent sites, thus suggesting indirect effects of kinase-phosphatase networks. This study provides a proof-of-concept of the use of real-time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.
authors.affiliation: Biomolecular NMR Laboratory, Department of Organic Chemistry, University of Barcelona, Baldiri Reixac, 10-12, 08028 Barcelona (Spain); Signaling and Cell Cycle Laboratory, Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona (Spain).
authors.forename: Irene
authors.initials: I
authors.lastname: Amata
authors.forename: Mariano
authors.initials: M
authors.lastname: Maffei
authors.forename: Ana
authors.initials: A
authors.lastname: Igea
authors.forename: Marina
authors.initials: M
authors.lastname: Gay
authors.forename: Marta
authors.initials: M
authors.lastname: Vilaseca
authors.forename: Angel R
authors.initials: AR
authors.lastname: Nebreda
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D014981:Xenopus / Q000254:growth & development / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D009587:Nitrogen Isotopes; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases
keywords: NMR spectroscopy; c-Src; intrinsically disordered proteins; mass spectrometry; phosphoproteins