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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.

Direct inhibitors of mutationally activated KRAS are currently under intense preclinical and clinical development, with one KRASG12C mutant-selective inhibitor approved. However, treatment-associated resistance to KRASG12C inhibitors has been reported, with ~60% of relapsed patients acquiring mutations in signaling components both upstream and downstream, and at the level of RAS itself, that led to reactivation of RAF-MEK-ERK and PI3K-AKT effector pathways. Unexpectedly, we found that ectopic expression of constitutively activated MEK1, ERK1 or ERK2, but not AKT1, drove near-complete resistance to direct inhibitors of KRASG12C or KRASG12D in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). Despite comprehensive studies, how ERK supports KRAS-dependent cancer growth remains poorly understood. Therefore, we focused on delineating the ERK-dependent phosphoproteome. We treated a panel of six KRAS-mutant PDAC cell lines acutely (1 h) and long-term (24 h) with the highly selective ERK1/2 inhibitor SCH772984. Our proteomics analyses identified 5,117 phosphosites on 2,252 proteins significantly dysregulated by ERK inhibition. We first compared our PDAC ERK phosphoproteome with a recent compendium of published direct/indirect ERK substrates identified in non-PDAC cells and found, surprisingly, only 12% overlap. Thus, our dataset substaintially extends the complexity of ERK-regulated phosphoproteins, in part, reflecting a pancreatic cancer selective profile. To elucidate the kinase network that drives our PDAC ERK-dependent phosphoproteome, we utilized our newly described global atlas of kinase-substrate specificities and reported kinase-substrate interaction datasets. This revealed a highly dynamic kinome dominated by ERK at 1 h, then dominated by ERK-regulation of CDK1-6 cell cycle components and RHO GTPase signaling. Next, we established the subcellular distribution of the PDAC ERK phosphoproteome, then evaluated data from DepMap, and found enrichment of nuclear ERK substrates that displayed strong dependencies in PDAC. We also determined the KRASG12C-regulated phosphoproteome in pancreatic, lung, and colorectal cancer cells using the KRASG12C selective inhibitor MRTX1257. We used our global atlas of kinase-motif specificities and ERK-regulated phosphoproteome to determine KRAS-regulated signaling pathways in KRASG12C mutant cancers. We found a high correlation between KRAS- and ERK-regulated phosphorylations, primarily driven by CDK substrates. However, we also noted key differences, principally in DNA damage response pathways not identified in our ERK-regulated phosphoproteome. Finally, we performed a high-throughput drug sensitivity and resistance screen of (DSRT) comprised of >500 clinically actionable drugs in combination with a KRASG12C inhibitor, and found ERK inhibitors as top hits. In summary, our studies establish a comprehensive profile of KRAS-ERK signaling output that may define new therapeutic targets for targeting KRAS- and ERK-dependent cancer growth. Citation Format: Jennifer E. Klomp, Jeff A. Klomp, Nathaniel J. Diehl, Cole A. Edwards, Kristina Drizyte-Miller, Priya S. Hibshman, Runying Yang, Alexis J. Morales, Khalilah E. Taylor, Mariaelena Pierobon, Emanuel F. Petricoin III, Johnson L. Jared, Emily M. Huntsman, Tomer M. Yaron, Markus Vähä-Koskela, Laura E. Herring, Alex W. Prevatte, Natalie K. Barker, Lee M. Graves, Wennerberg Krister, Lewis C. Cantley, James G. Christensen, Adrienne D. Cox, Channing J. Der, Clint A. Stalnecker. Determination of KRAS- and ERK-regulated phosphoproteomes in KRAS-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B034.

Numerous physiological processes are governed by tyrosine phosphorylation. The protein tyrosine kinases that carry this out are dysregulated in a variety of cancers and are the targets of efficacious drugs. For most tyrosine kinases, we have a very limited understanding of their downstream signaling pathways and phosphorylation targets. Conversely, thousands of tyrosine phosphorylation events have so far been experimentally identified, and the overwhelming majority of these do not have an associated kinase. In this work, we have utilized peptide arrays to determine the substrate sequence specificity for the human tyrosine kinome, comprising 46 receptor tyrosine kinases, 32 nonreceptor tyrosine kinases, and 14 noncanonical tyrosine kinases. Nearly every kinase we profiled favored tyrosines that were positioned next to phosphorylated amino acids, indicating that PTM priming and crosstalk are general properties of the tyrosine kinome. Comparisons with C. elegans orthologs suggested that tyrosine kinase substrate motifs have remained conserved throughout metazoan evolution. When we computationally utilized our dataset to identify kinases for tyrosine substrates, our predictions were in strong accordance with published reports. When applied to high-throughput tyrosine phosphoproteomics datasets, we could decipher the kinases involved in cellular responses to growth factors, oncogenic mutations, and targeted inhibitors. Together, this work uncovers fundamental rules of substrate selection for nearly the entire human tyrosine kinome and provides a comprehensive resource for connecting tyrosine phosphorylation events with their kinases and signaling pathways. Citation Format: Jared L. Johnson, Tomer M. Yaron, Lewis C. Cantley, Michael B. Yaffe, Benjamin E. Turk, Emily M. Huntsman, Peter V. Hornbeck. An atlas of substrate specificities for the human tyrosine kinome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3487.

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.

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 error 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 which binds to TBK1 on the centrosomes to activate it. 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 also are 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 at S318 promoting its degradation by ubiquitin proteasomal system acting as a negative regulatory step. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.

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.