cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R(2)C(2) holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

ONE SENTENCE SUMMARY A Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2 in vitro and lead to rapid on-target degradation of TRIB2 in human cancer cells. SHORT ABSTRACT Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase ‘inhibitors’, which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and on-target TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules. FULL ABSTRACT A major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2. Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2 in vitro . Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug ‘off-targets’ might be relevant for other inhibitors that serendipitously target pseudokinases. ABBREVIATIONS DSF Differential Scanning Fluorimetry EGFR Epidermal Growth Factor Receptor HER2 Human Epidermal Growth Factor Receptor 2 MS Mass spectrometry MST MicroScale Thermophoresis PKIS Published Kinase Inhibitors Set TRIB2 Tribbles 2 TSA Thermal Stability Assa

Correction: Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

[This corrects the article DOI: 10.1371/journal.pgen.1005885.]

Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases

Operation Moonshot: rapid translation of a SARS-CoV-2 targeted peptide immunoaffinity liquid chromatography-tandem mass spectrometry test from research into routine clinical use

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory

Plk4 and Aurora A cooperate in the initiation of acentriolar spindle assembly in mammalian oocytes

Establishing the bipolar spindle in mammalian oocytes after their prolonged arrest is crucial for meiotic fidelity and subsequent development. In contrast to somatic cells, the first meiotic spindle assembles in the absence of centriole-containing centrosomes. Ran-GTP can promote microtubule nucleation near chromatin, but additional unidentified factors are postulated for the activity of multiple acentriolar microtubule organizing centers in the oocyte. We now demonstrate that partially overlapping, nonredundant functions of Aurora A and Plk4 kinases contribute to initiate acentriolar meiosis I spindle formation. Loss of microtubule nucleation after simultaneous chemical inhibition of both kinases can be significantly rescued by drug-resistant Aurora A alone. Drug-resistant Plk4 can enhance Aurora A–mediated rescue, and, accordingly, Plk4 can phosphorylate and potentiate the activity of Aurora A in vitro. Both kinases function distinctly from Ran, which amplifies microtubule growth. We conclude that Aurora A and Plk4 are rate-limiting factors contributing to microtubule growth as the acentriolar oocyte resumes meiosis.L. Bury was the recipient of a Cancer Research UK research studentship from Cambridge Cancer Centre. P.A. Coelho is supported by Cancer Research UK program grant C3/A18795 to D.M. Glover. M. Zernicka-Goetz is a Wellcome Trust Senior Fellow. P.A. Eyers acknowledges North West Cancer Research for additional support (grants CR1037 and CR1088)

Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high-throughput phosphoproteomics. Here we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. In addition to evaluating multiple fragmentation regimes (HCD, EThcD, and neutral-loss-triggered ET­(ca/hc)­D) and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with <i>ptmRS</i> for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high-resolution orbitrap-based MS/MS analysis, employing MASCOT/<i>ptmRS</i> for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlight that a charge-state-dependent fragmentation regime and a multiple algorithm search strategy are likely to be of benefit for confident large-scale phosphosite localization