Phosphorylation network rewiring by gene duplication

Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post‐translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post‐duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase–substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post‐translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them.     

Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics

The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase‐regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH, Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.     

Protein kinases phosphorylate long disordered regions in intrinsically disordered proteins

Phosphorylation is a major post‐translational modification that plays a central role in signaling pathways. Protein kinases phosphorylate substrates (phosphoproteins) by adding phosphate at Ser/Thr or Tyr residues (phosphosites). A large amount of data identifying and describing phosphosites in phosphoproteins has been reported but the specificity of phosphorylation is not fully resolved. In this report, data of kinase‐substrate pairs identified by the Kinase‐Interacting Substrate Screening (KISS) method were used to analyze phosphosites in intrinsically disordered regions (IDRs) of intrinsically disordered proteins. We compared phosphorylated and nonphosphorylated IDRs and found that the phosphorylated IDRs were significantly longer than nonphosphorylated IDRs. The phosphorylated IDR is often the longest IDR (71%) in a phosphoprotein when only a single phosphosite exists in the IDR, and when the phosphoprotein has multiple phosphosites in an IDR(s), the phosphosites are primarily localized in a single IDR (78%) and this IDR is usually the longest one (81%). We constructed a stochastic model of phosphorylation to estimate the effect of IDR length. The model that accounted for IDR length produced more realistic results when compared with a model that excluded the IDR length. We propose that the IDR length is a significant determinant for locating kinase phosphorylation sites in phosphoproteins.     

Time Course of Changes in Sorafenib‐Treated Hepatocellular Carcinoma Cells Suggests Involvement of Phospho‐Regulated Signaling in Ferroptosis Induction

Ferroptosis is a form of regulated, non‐apoptotic cell death characterized by excessive lipid peroxidation that can be triggered by inhibition of the cystine‐glutamate antiporter, system X_c^?. Sorafenib, an FDA‐approved multi‐kinase inhibitor drug that is used for treatment of hepatocellular carcinoma (HCC), has been shown to induce ferroptosis. Protein phosphorylation changes upon sorafenib treatment have been previously reported in patient studies and in cell culture. However, early phosphorylation changes during induction of ferroptosis are not reported. This work highlights these changes through a time course from 7 to 60 min of sorafenib treatment in human (SKHep1) HCC cells. A total of 6170 unique phosphosites from 2381 phosphoproteins are quantified, and phosphorylation changes occur after as little as 30 min of sorafenib treatment. By 60 min, notable changes included phosphosites significantly changing on p53 (P04637), CAD protein (P27708), and proteins important for iron homeostasis, such as heavy chain ferritin (FTH1; P02794), heme oxygenase 1 (HMOX1; P09601), and PCBP1 (Q15365). Additional sites on proteins in key regulatory pathways are identified, including sites in ferroptosis‐related proteins, indicating the likely involvement of phospho‐regulated signaling during ferroptosis induction.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Serine and threonine residues of plant STN7 kinase are differentially phosphorylated upon changing light conditions and specifically influence the activity and stability of the kinase

STN7 kinase catalyzes the phosphorylation of the globally most common membrane proteins, the light‐harvesting complex II (LHCII) in plant chloroplasts. STN7 itself possesses one serine (Ser) and two threonine (Thr) phosphosites. We show that phosphorylation of the Thr residues protects STN7 against degradation in darkness, low light and red light, whereas increasing light intensity and far red illumination decrease phosphorylation and induce STN7 degradation. Ser phosphorylation, in turn, occurs under red and low intensity white light, coinciding with the client protein (LHCII) phosphorylation. Through analysis of the counteracting LHCII phosphatase mutant tap38/pph1, we show that Ser phosphorylation and activation of the STN7 kinase for subsequent LHCII phosphorylation are heavily affected by pre‐illumination conditions. Transitions between the three activity states of the STN7 kinase (deactivated in darkness and far red light, activated in low and red light, inhibited in high light) are shown to modulate the phosphorylation of the STN7 Ser and Thr residues independently of each other. Such dynamic regulation of STN7 kinase phosphorylation is crucial for plant growth and environmental acclimation.     

Scaffold proteins in mammalian MAP kinase cascades

The mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.     

Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1

The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal–regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of protein kinases in signaling modules. In addition to the well-documented T202 and Y204 stimulatory phosphorylation sites in the activation T-loop of ERK1 and its closest relative, ERK2, three additional flanking phosphosites have been confirmed (T198, T207, and Y210 from ERK1) by high-throughput mass spectrometry. In vitro kinase assays revealed the functional importance of T207 and Y210, but not T198, in negatively regulating ERK1 catalytic activity. The Y210 site could be important for proper conformational arrangement of the active site, and a Y210F mutant could not be recognized by MEK1 for phosphorylation of T202 and Y204 in vitro. Autophosphorylation of T207 reduces the catalytic activity and stability of activated ERK1. We propose that after the activation of ERK1 by MEK1, subsequent slower phosphorylation of the flanking sites results in inhibition of the kinase. Because the T207 and Y210 phosphosites of ERK1 are highly conserved within the eukaryotic protein kinase family, hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases.     

Inducing autophagy

Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR activity was not affected, but indicated increased MAPK3 activity, regulation of proteins involved in Rho signal transduction, and a novel phosphorylation motif, serine-proline-threonine (SPT), which could be linked to cytoskeleton-associated proteins. MAPK3 could not be identified as the primary driver of ammonia-induced autophagy but instead the data suggested an upregulation of AMPK and the unfolded protein response (UPR), which might link ammonia to autophagy induction. Support of UPR induction was further obtained from the finding of increased protein levels of the ER stress markers DDIT3/CHOP and HSPA5 during ammonia treatment. The large-scale data set presented here comprises extensive high-quality quantitative information on phosphoprotein regulation in response to 2 very different autophagy inducers and should therefore be considered a general resource for the community.     

Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

Protein phosphorylation plays a critical role in the regulation and progression of mitosis. >40,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4100 and > 2200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.     

Phosphoproteomic analyses reveal novel cross‐modulation mechanisms between two signaling pathways in yeast

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen‐activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time‐resolved phosphorylation site dynamics after pathway co‐stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone‐induced down‐regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.     

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.     

Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo

In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo. Here, we used an automated, robust, and sensitive online TiO 2-based LC-MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approximately 180 microg), 321 from 10 embryos, and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This data set, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA, and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide data set was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several proteins suggesting preserved function in embryonic development.     

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR‐90 cells infected with human adenovirus type 2 (Ad2) is used in a time‐course quantitative analysis combining titanium dioxide (TiO₂) particle enrichment and SILAC‐MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP‐dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post‐infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus‐induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post‐infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.     

Profiling of Histidine Phosphoproteome in Danio rerio by TiO2 Enrichment

Histidine phosphorylation is a reversible post‐translational modification that is known to regulate signal transduction in prokaryotes. However, functional studies in eukaryotes have been largely neglected due to the labile nature of N‐linked phosphorylated amino acids. In an effort to help elucidate the heretofore hidden vertebrate phosphoproteome, this report presents a global phosphorylation analysis of Danio rerio (zebrafish) larvae. Phosphopeptide enrichment is performed using a TiO₂ affinity technique. A total of 68 unique phosphohistidine sites are detected on 63 proteins among 1076 unique phosphosites on 708 proteins. Data are available via ProteomeXchange with identifier PXD012735. This report provides the first phosphohistidine dataset obtained from zebrafish.     

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.     

In silico studies of potential phosphoresidues in the human nucleophosmin/B23: its kinases and related biological processes

Human nucleophosmin/B23 is a phosphoprotein involved in ribosome biogenesis, centrosome duplication, cancer, and apoptosis. Its function, localization, and mobility within cells, are highly regulated by phosphorylation events. Up to 21 phosphosites of B23 have been experimentally verified even though the corresponding kinase is known only for seven of them. In this work, we predict the phosphorylation sites in human B23 using six kinase-specific servers (KinasePhos 2.0, PredPhospho, NetPhosK 1.0, PKC Scan, pkaPS, and MetaPredPS) plus DISPHOS 1.3, which is not kinase specific. The results were integrated with information regarding 3D structure and residue conservation of B23, as well as cellular localizations, cellular processes, signaling pathways and protein-protein interaction networks involving both B23 and each predicted kinase. Thus, all 40 potential phosphosites of B23 were predicted with significant score (>0.50) as substrates of at least one of 38 kinases. Thirteen of these residues are newly proposed showing high susceptibility of phosphorylation considering their solvent accessibility. Our results also suggest that the enzymes CDKs, PKC, CK2, PLK1, and PKA could phosphorylate B23 at higher number of sites than those previously reported. Furthermore, PDK, GSK3, ATM, MAPK, PKB, and CHK1 could mediate multisite phosphorylation of B23, although they have not been verified as kinases for this protein. Finally, we suggest that B23 phosphorylation is related to cellular processes such as apoptosis, cell survival, cell proliferation, and response to DNA damage stimulus, in which these kinases are involved. These predictions could contribute to a better understanding, as well as addressing further experimental studies, of B23 phosphorylation.