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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.

In this study, Wu et al. set out to understand the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Using a ubiquitin binding domain protein array screen, they identify Cezanne and Cezanne2 as two new components in the complex ubiquitin landscape at DNA damage sites for regulating recruitment of DNA repair and signaling proteins. They further show that Cezanne serves as a “reader” of the Lys63-linkage localizing to DNA damage sites and an “eraser” of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure–affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques—Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance—were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein–ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1–S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631–633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.

We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a Kdof ∼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.

A phosphoswitch in the tyrosine phosphatase PTPROt determines whether it stimulates or inhibits the kinase Src.