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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.

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.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

In response to genotoxic stress, multiple kinase signaling cascades are activated, many of them directed towards the tumor suppressor p53, which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10, and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation by modulating the p53-dependent transcriptional output. NEK10 directly phosphorylates p53 on Y327, revealing NEK10's unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA-damaging agents. Further, a combinatorial score of NEK10 and TP53-target gene expression is an independent predictor of a favorable outcome in breast cancers.

Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems., Author summary Protein kinases, which catalyze the transfer of phosphate from ATP to substrate proteins, are important enzymes in cellular signal transduction pathways mediating responses to extracellular cues. In order to function properly in signal transmission, each kinase must phosphorylate only a limited number of proteins among the thousands present within the cell. One way that kinases choose their substrates is by recognition of amino-acid sequence motifs surrounding the site of phosphorylation, but kinases can also recruit substrates through docking interactions occurring outside of the catalytic cleft. For most kinases, the relative contribution of catalytic site and docking site interactions to substrate selection is not known. Here, we investigated the phosphorylation site specificity of a group of 30 human protein serine–threonine kinases called the STE20 family. Guided by x-ray crystal structures of kinase-peptide complexes, we re-engineered the catalytic clefts of two members of this group, PAK4 and MST4, to exchange their phosphorylation site motifs. In cells, the re-engineered form of PAK4 was unable to phosphorylate substrates of wild-type PAK4 or to carry out its normal function in reorganizing the actin cytoskeleton. In contrast, it was able to function in place of MST4 and related kinases in the growth-controlling Hippo signaling pathway. Overall, these studies suggest that catalytic site interactions can be both necessary and sufficient for kinase function.

Although elevated levels of reactive oxygen species (ROS) have been observed in cancer cells and cancer cells aberrantly proliferate, it is not known whether the level of reactive oxygen species and the accumulation of oxidative damage to macromolecules vary across the cell cycle. Here, we measure the prevalence of reactive oxygen species and of biomolecule oxidation across the cell cycle in freely cycling cancer cells. We report that reactive oxygen species vary during the cell cycle and peak in mitosis, resulting in mitotic accumulation of oxidized protein cysteine residues. Prolonged mitotic arrest further increased the levels of ROS and the abundance of oxidatively damaged biomolecules, including cysteine-sulfenic-acid-containing proteins and 8-oxoguanine. These finding suggest that mitotic arrest agents may enhance the effects of ROS-dependent anticancer therapies. We studied connections between ROS and the cell cycle in unsynchronized cancer cells and using multiple independent assays found that ROS and oxidative damage to biomolecules are highest in mitosis and can be further enhanced by mitotic arrest., National Institutes of Health (U.S.) (Grant R01-GM104047), National Institutes of Health (U.S.) (Grant R01-ES015339), National Institutes of Health (U.S.) (Grant R35-ES028374), National Institutes of Health (U.S.) (Grant U54-CA112967), National Institutes of Health (U.S.) (Grant U54-CA217377), United States. Department of Defense. Peer-Reviewed Medical Research Program (Contract W81XWH-16-1-0464), Koch Institute Support Grant (P30-CA14051), Center for Environmental Health and Injury Control (U.S.) (Support Grant P30-ES002109)

In response to genotoxic stress, multiple kinase signalling cascades are activated, many of them directed towards the tumour suppressor p53 which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10 and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation through modulation of the p53-dependent transcriptional output, by directly phosphorylating p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA damaging agents and low NEK10 expression is an independent predictor of a favorable response to radiation treatment in WT TP53 breast cancer patients.

Human NimA-related kinases (Neks) have multiple mitotic and non-mitotic functions, but few substrates are known. We systematically determined the phosphorylation-site motifs for the entire Nek kinase family, except for Nek11. While all Nek kinases strongly select for hydrophobic residues in the −3 position, the family separates into four distinct groups based on specificity for a serine versus threonine phospho-acceptor, and preference for basic or acidic residues in other positions. Unlike Nek1-Nek9, Nek10 is a dual-specificity kinase that efficiently phosphorylates itself and peptide substrates on serine and tyrosine, and its activity is enhanced by tyrosine auto-phosphorylation. Nek10 dual-specificity depends on residues in the HRD+2 and APE-4 positions that are uncommon in either serine/threonine or tyrosine kinases. Finally, we show that the phosphorylation-site motifs for the mitotic kinases Nek6, Nek7 and Nek9 are essentially identical to that of their upstream activator Plk1, suggesting that Nek6/7/9 function as phospho-motif amplifiers of Plk1 signaling.

All 6 human prosurvival Bcl-2 proteins can drive cancer development and contribute to therapy resistance. However, their relative abilities to protect cells against cancer therapy were not examined previously. We report that Bcl-2, Bcl-xL, or Bcl-w consistently protected leukemic cells better than Bcl-B, Bfl-1, or Mcl-1 against a wide variety of anticancer regimens. Current thinking would attribute this to differences in their ability to bind to BH3-only proteins, Bax, and Bak. To address this, we established the first complete, quantitative cellular interaction profile of all human prosurvival Bcl-2 proteins with all their proapoptotic relatives. Binding was unexpectedly promiscuous, except for Bad and Noxa, and did not explain the differential antiapoptotic capacity of the Bcl-2 proteins. Rather, Bcl-B, Bfl-1, or Mcl-1 proved less potent due to steady-state or drug-induced proteasomal degradation. All 6 Bcl-2 proteins similarly protected against the diverse anticancer regimens when expressed at equal protein levels, in agreement with their broad interaction profile. Therefore, clinical diagnostics should include all family members and should be performed at the protein rather than at the messenger RNA level. In drug development, targeting the ubiquitination machinery of prosurvival Bcl-2 proteins will complement and potentially improve on targeting Bcl-2 protein interactions with BH3 mimetics.

Summary During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term “minority MOMP.” Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis., Graphical Abstract, Highlights • MOMP can occur in a minority of mitochondria • Minority MOMP triggers caspase activity but fails to kill cells • Minority MOMP-induced caspase activity causes DNA damage and genomic instability • Minority MOMP promotes cellular transformation and tumorigenesis, During apoptosis, mitochondrial outer membrane permeabilization (MOMP) is widespread, leading to rapid cell death. Here, Ichim et al. demonstrate that MOMP can also be engaged in a minority of mitochondria without killing the cell. Instead, minority MOMP triggers caspase-dependent DNA damage and genomic instability, thereby promoting transformation and tumorigenesis.

Background: Endogenous functions of the recently identified membrane-associated RING-CH (MARCH) family of transmembrane ubiquitin ligases are undefined, except for MARCH-1. Results: MARCH-8 interacts with death receptor TRAIL receptor (R) R1, ubiquitinates TRAIL-R1, and down-regulates TRAIL-R1 from the cell surface at steady state. Conclusion: Steady-state cell surface levels of TRAIL-R1 are controlled by MARCH-8-mediated ubiquitination. Significance: TRAIL-R1 is the first identified substrate of endogenous MARCH-8., The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.