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While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage., Competing Interests: Declaration of interests R.G. is a member of the scientific advisory board (SAB) of Alltrna, Initial Therapeutics, and Arrakis Pharmaceuticals, consults for Vertex Pharmaceuticals, Brystol-Myers Squibb (Celgene), Monta Rosa Therapeutics, and Flagship Pioneering, and served on the SAB at Moderna. A.O. received consulting fees from Nine Square Therapeutics Co. L.C.C. is founder and member of the board of directors of Agios Pharmaceuticals; is founder and receives support from Petra Pharmaceuticals; is listed as inventor on a patent (WO2019232403A1, Weill Cornell Medicine); is co-founder and shareholder in Faeth Therapeutics; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur, and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. T.M.Y.-B. is a co-founder of DeStroke. J.L.J received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. R.G., S.R., N.K.S., and C.M. are listed as inventors on U.S. Provisional Patent Application No. 63/652,824, filed May 29, 2024., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur., Competing Interests: Conflict of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. P. G. is a member of the scientific advisory boards of Cell Signaling Technologies and ThermoFisher Scientific. 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; is listed as an inventor on a patent (WO2019232403A1, Weill Cornell Medicine) for combination therapy for PI3K-associated disease or disorder, and the identification of therapeutic interventions to improve response to PI3K inhibitors for cancer treatment; is a co-founder and shareholder in Faeth Therapeutics; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J. L. J has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T. M. Y.-B. is a co-founder of DeStroke., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth 1 . Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome 1-3 . How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood 4-7 . 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., (© 2024. The Author(s).)

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents., Competing Interests: Declaration of interests R. Sebra is currently a paid consultant and equity holder at GeneDx. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals; is a founder and receives research support from Petra Pharmaceuticals; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur, and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. T.M.Y. is a co-founder and stockholder of DeStroke., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling., Competing Interests: Declaration of interests N.V. reports consultant and advisory board activities for Novartis, Petra Pharmaceuticals, Reactive Biosciences, and Magnet Biomedicine. E.A.K. is a shareholder of Eli Lilly and Company, and E.A.K. and K.K. are employees of Loxo Oncology at Lilly. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals; is a founder and receives research support from Petra Pharmaceuticals; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur, and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. J.L.J. reports consultant activities for Scorpion Therapeutics and Volastra Therapeutics. M.D.G. reports personal fees from Novartis AG, Pfizer, Inc., and Scorpion Therapeutics. L.C.C., B.D.H., and M.D.G. are inventors on patents for Combination Therapy for PI3K-associated Disease or Disorder and The Identification of Therapeutic Interventions to Improve Response to PI3K Inhibitors for Cancer Treatment. B.D.H., L.C.C., and M.D.G. are co-founders and shareholders in Faeth Therapeutics. R.E.S. is on the sponsored advisory board for Miromatrix, Inc., and Lime Therapeutics and is a consultant and speaker for Alnylam, Inc. T.M.Y. is a stockholder and on the board of directors of DESTROKE, Inc., an early-stage start-up developing mobile technology for automated clinical stroke detection., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer driver events refer to key genetic aberrations that drive oncogenesis; however, their exact molecular mechanisms remain insufficiently understood. Here, our multi-omics pan-cancer analysis uncovers insights into the impacts of cancer drivers by identifying their significant cis-effects and distal trans-effects quantified at the RNA, protein, and phosphoprotein levels. Salient observations include the association of point mutations and copy-number alterations with the rewiring of protein interaction networks, and notably, most cancer genes converge toward similar molecular states denoted by sequence-based kinase activity profiles. A correlation between predicted neoantigen burden and measured T cell infiltration suggests potential vulnerabilities for immunotherapies. Patterns of cancer hallmarks vary by polygenic protein abundance ranging from uniform to heterogeneous. Overall, our work demonstrates the value of comprehensive proteogenomics in understanding the functional states of oncogenic drivers and their links to cancer development, surpassing the limitations of studying individual cancer types., Competing Interests: Declaration of interests Y.G. is a consultant for Oriel Research Therapeutics. T.M.Y. is a co-founder, stockholder, and member of the board of directors of DESTROKE, Inc., an early-stage start-up developing mobile technology for automated clinical stroke detection. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. F.A. is an inventor on a patent application related to SignatureAnalyzer-GPU and has been an employee of Illumina, Inc., since 8 November 2021. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals and is a founder of 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. G.G. receives research funds from IBM, Pharmacyclics, and Ultima Genomics and is also an inventor on patent applications filed by the Broad Institute related to MSMuTect, MSMutSig, POLYSOLVER, SignatureAnalyzer-GPU, MSIDetect, and MinimuMM-Seq. G.G. is also a founder, consultant, and privately held equity in Scorpion Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2023. The Author(s).)

The microsporidia Nosema ceranae is an obligate intracellular parasite that causes honey bee mortality and contributes to colony collapse. Fumagillin is presently the only pharmacological control for N. ceranae infections in honey bees. Resistance is already emerging, and alternative controls are critically needed. Nosema spp. exhibit increased sensitivity to heat shock, a common proteotoxic stress. Thus, we hypothesized that targeting the Nosema proteasome, the major protease removing misfolded proteins, might be effective against N. ceranae infections in honey bees. Nosema genome analysis and molecular modeling revealed an unexpectedly compact proteasome apparently lacking multiple canonical subunits, but with highly conserved proteolytic active sites expected to be receptive to FDA-approved proteasome inhibitors. Indeed, N. ceranae were strikingly sensitive to pharmacological disruption of proteasome function at doses that were well tolerated by honey bees. Thus, proteasome inhibition is a novel candidate treatment strategy for microsporidia infection in honey bees.

In other species characterized to date, aging, as a function of reproductive potential, results in the breakdown of proteaostasis and a decreased capacity to mount responses by the heat shock response (HSR) and other proteostatic network pathways. Our understanding of the maintenance of stress pathways, such as the HSR, in honey bees, and in the reproductive queen in particular, is incomplete. Based on the findings in other species showing an inverse relationship between reproductive potential and HSR function, one might predict that that HSR function would be lost in the reproductive queens. However, as queens possess an atypical uncoupling of the reproduction-maintenance trade-off typically found in solitary organisms, HSR maintenance might also be expected. Here we demonstrate that reproductive potential does not cause loss of HSR performance in honey bees as queens induce target gene expression to levels comparable to those induced in attendant worker bees. Maintenance of HSR function with advent of reproductive potential is unique among invertebrates studied to date and provides a potential model for examining the molecular mechanisms regulating the uncoupling of the reproduction-maintenance trade-off in queen bees, with important consequences for understanding how stresses impact different types of individuals in honey bee colonies.