3 research outputs found
An atlas of substrate specificities for the human serine/threonine kinome
Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.</p
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The intrinsic substrate specificity of the human tyrosine kinome.
Acknowledgements: We thank M. J. Begley, F. M. White, G. Getz, S. R. Hubbard, N. Shah and M. L. Hemming for discussions; and Y. Ma, M. R. Lundquist, K. Liberatore, T. M. Levy, S. A. Beausoleil, J. Wong, S. Petovic, M. Tran and the staff at Signalchem Biotech for technical assistance. T.M.Y.-B. thanks D. Yaron-Barir, S. Yaron, N. Yaron, J. R. Haddad and S. Haddad for their support. J.L.J. thanks M. Bak-Johnson, C. Ahn, S. Bak, J. W. Erickson and R. A. Cerione for their support. This research was supported by Leukemia & Lymphoma Society Award (to J.L.J. and L.C.C.); the Claudia Adams Barr Program for Cancer Research Award (to J.L.J.); National Institute of Health grants P01 CA120964 (to L.C.C.), R35-CA197588 (to L.C.C.), P01-CA117969 (to L.C.C.), R35-ES028374 (to M.B.Y.), R01-CA226898 (to M.B.Y.), R01-GM135331 (to B.E.T.) and R01-GM104047 (to B.E.T. and M.B.Y.); the joint Cancer Research UK and Brain Tumour Charity funded Brain Tumour Award C42454/A28596 (to M.B.Y.); the Charles and Marjorie Holloway Foundation (to M.B.Y.); the MIT Center for Precision Cancer Medicine (to M.B.Y.); the Jane Coffin Childs Memorial Fund (to J.M.O.); the Howard Hughes Medical Institute Hanna H. Gray Fellow award (to J.M.O.); and Cancer Research UK grants C9685/A26398 (to P.C.) and C9545/A29580 (to P.C.).Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution
An atlas of substrate specificities for the human serine/threonine kinome
Abstract Protein phosphorylation is one of the most widespread post-translational modifications in biology 1,2 . With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes 3,4 . For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible 3 . Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways