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.
Display omitted
•Immediate-early response to UV is dominated by ribosome-mediated signaling•UV-induced apoptosis is mediated by ZAK, not the DNA damage response pathway•GCN2 regulates ZAK-mediated apoptosis by limiting ribosome collisions•ZAK degradation limits apoptosis by rendering cells tolerant to ribotoxic stress
A comprehensive analysis of molecular and cellular changes following UV irradiation reveals that the ribotoxic stress response, rather than the DNA damage response, mediates UV-dependent programmed cell death.
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 TMT 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.
Display omitted
•T cells undergo significant proteome, phosphoproteome, and protein thermal stability remodeling during activation.•The phosphorylation-integrated thermal shift assay combines protein and phosphoprotein abundance with thermal stability measurements in a single TMT-plex.•DNA repair pathways are altered in protein and phosphorylation site abundance, and protein thermal stability during T cell activation.•Changes in cyclin-dependent kinase activity correlate with changes in CDK substrate thermal stability.
T cells undergo a significant rearrangement of their proteome and phosphoproteome during activation while accumulating necessary cellular components for rapid cell division. Here, we describe the phosphorylation integrated thermal shift assay, which combines the measurement of protein and phosphorylation site abundance with the measurement of protein and phosphorylation site thermal stability. This assay may help determine which proteome and phosphoproteome changes are functionally relevant in T cell activation by identifying proteins/phosphorylation sites with altered biophysical states, and presumably altered function.
Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth
. Multicellular eukaryotes typically have more than 50 distinct ...protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome
. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood
. 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.
CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these ...tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on
Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.
Although genomic anomalies in glioblastoma (GBM) have been well studied for over a decade, its 5-year survival rate remains lower than 5%. We seek to expand the molecular landscape of high-grade ...glioma, composed of IDH-wildtype GBM and IDH-mutant grade 4 astrocytoma, by integrating proteomic, metabolomic, lipidomic, and post-translational modifications (PTMs) with genomic and transcriptomic measurements to uncover multi-scale regulatory interactions governing tumor development and evolution. Applying 14 proteogenomic and metabolomic platforms to 228 tumors (212 GBM and 16 grade 4 IDH-mutant astrocytoma), including 28 at recurrence, plus 18 normal brain samples and 14 brain metastases as comparators, reveals heterogeneous upstream alterations converging on common downstream events at the proteomic and metabolomic levels and changes in protein-protein interactions and glycosylation site occupancy at recurrence. Recurrent genetic alterations and phosphorylation events on PTPN11 map to important regulatory domains in three dimensions, suggesting a central role for PTPN11 signaling across high-grade gliomas.
Display omitted
•Metabolome and glycoproteome data reveal driver interactions and recurrence markers•Alterations in TERTp, PTEN, or TERTp/EGFR produce similar molecular features•PTPN11 signaling links EGFR, PDGFR, and IDH1 to downstream effectors•A low hypoxia signature and reduced AMPKA activities are found in IDH-mutant HGG
Liu et al. integrate 14 proteogenomic, glycoproteomic, and metabolomic platforms on 228 high-grade gliomas composed of glioblastomas and grade 4 IDH-mutant astrocytomas, including tumors at recurrence. This study identifies multi-scale regulatory interactions governing tumor development and evolution, reveals heterogeneous upstream alterations converging on common downstream events, and suggests a central role for PTPN11 signaling across high-grade gliomas.
