A global atlas of substrate specificities for the human serine/threonine kinome

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.

High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display

“…If so, this would be a potentially powerful tool for the identification of native substrates and the dissection of phosphotyrosine signaling pathways. Indeed, oriented peptide libraries have been applied extensively to predict the native substrates of protein kinases (Johnson et al, 2022; Miller et al, 2008; Obenauer et al, 2003). We are particularly interested in using high-throughput specificity screens to predict how mutations proximal to phosphorylation sites affect tyrosine kinase selectivity.…”

“…The specificity profiling screens described thus far were constrained to sequences that contain the canonical twenty amino acids. Several studies have suggested that non-canonical amino acids and post-translationally modified amino acids can also impact sequence recognition by tyrosine kinases and SH2 domains (Alfaro-Lopez et al, 1998; Begley et al, 2015; Chapelat et al, 2012; Johnson et al, 2022; Yeh et al, 2001). The most notable example of this is phospho-priming, whereby phosphorylation of one residue on a protein enhances the ability of a kinase to recognize and phosphorylate a proximal residue.…”

“…Several variations on this technique have been reported to improve the throughput and quantification of sequence preferences (Deng et al, 2014; Huang et al, 2008; Hutti et al, 2004; Mok et al, 2010). Notably, this method is also applicable to serine/threonine kinases, and large swaths of the yeast and human kinomes have been characterized using oriented peptide libraries, providing significant insights into kinase-substrate recognition and phospho-signaling (Deng et al, 2014; Johnson et al, 2022; Mok et al, 2010; Songyang et al, 1995). Oriented peptide library screens have primarily been useful for determining the preference for each amino acid at a given position, independent of sequence context, but evidence suggests that some amino acid preferences may depend on the surrounding sequence (Cantor et al, 2018).…”

Phosphoproteomics data-driven signalling network inference: does it work?

“…This corroborates the value of introducing a sequence similarity-based feature in network inference strategies and highlights the importance of reducing the noise in a dataset before applying network inference methods. In addition, efforts have recently been made to identify the substrate recognition motifs for all kinases 43,44 , which will further improve our ability to reduce noise and match kinases to the correct substrates. Additional ways to reduce noise in the dataset could include filters to include phosphosites that are predicted to be functional 45 , and introducing very strict p-value and fold-change cut-offs for the identification of differentially abundant phosphosites.…”

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases

“…Numerous mechanisms can underlie signaling specificity by protein kinases, including subcellular compartmentalization, scaffolding, signal dynamics, and direct kinase-substrate interactions 6 . One mechanism common to Ser/Thr protein kinases involves the recognition of sequence motifs surrounding sites of phosphorylation by the catalytic cleft 7 . However, since all MAPKs target a common Ser/Thr-Pro consensus sequence, catalytic site specificity is insufficient to mediate selectivity for a particular member of the family.…”