ABSTRACT
Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling 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 full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.
Competing Interest Statement
J.L.J has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y. is a co-founder, stockholder and on the board of directors of DESTROKE, Inc., an early-stage start-up developing mobile technology for automated clinical stroke detection. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals. L.C.C. is an inventor on patents (pending) for Combination Therapy for PI3K-associated Disease or Disorder, and The Identification of Therapeutic Interventions to Improve Response to PI3K Inhibitors for Cancer Treatment. L.C.C. is a co-founder and shareholder in Faeth Therapeutics. O.E. is a founder and equity holder of Volastra Therapeutics and OneThree Biotech. O.E. is a member of the scitific advisory board of Owkin, Freenome, Genetic Intelligence, Acuamark and Champions Oncology. O.E. receives research support from Eli Lilly, Janssen and Sanofi. D.J.T. is a member of the scientific advisory board at Dewpoint Therapeutics. A.D. is an equity holder of Denali Therapeutics. A.D. receives research support from Interline Therapeutics. N.V. reports consulting activities for Novartis and is on the scientific advisory board of Heligenics. M.D.G. is a co-founder and shareholder of Faeth Therapeutics, which are developing dietary and pharmacologic therapies for cancer. M.D.G. has received speaking and/or consulting fees from Pfizer Inc., Novartis AG, Scorpion Therapeutics, and Faeth Therapeutics.