Identifications of Putative PKA Substrates with Quantitative Phosphoproteomics and Primary-Sequence-Based Scoring.

Protein kinase A (PKA or cAMP-dependent protein kinase) is a serine/threonine kinase that plays essential roles in the regulation of proliferation, differentiation, and apoptosis. To better understand the functions of PKA, it is necessary to elucidate the direct interplay between PKA and their substrates in living human cells. To identify kinase target substrates in a high-throughput manner, we first quantified the change of phosphoproteome in the cells of which PKA activity was perturbed by drug stimulations. LC-MS/MS analyses identified 2755 and 3191 phosphopeptides from experiments with activator or inhibitor of PKA. To exclude potential indirect targets of PKA, we built a computational model to characterize the kinase sequence specificity toward the substrate target site based on known kinase-substrate relationships. Finally, by combining the sequence recognition model with the quantitative changes in phosphorylation measured in the two drug perturbation experiments, we identified 29 reliable candidates of PKA targeting residues in living cells including 8 previously known substrates. Moreover, 18 of these sites were confirmed to be site-specifically phosphorylated in vitro. Altogether this study proposed a confident list of PKA substrate candidates, expanding our knowledge of PKA signaling network.

Analytical strategies for shotgun phosphoproteomics: status and prospects.

New analytical strategies for phosphoproteomics, both experimental and computational, have been rapidly introduced in recent years, leading to novel biological findings on the role of protein phosphorylation, which have in turn stimulated further development of the analytical techniques. In this review, we describe the development of analytical strategies for LC-MS/MS-based phosphoproteomics, focusing particularly on recent progress in phosphopeptide enrichment, LC-MS/MS measurement and the subsequent computational analysis. High-coverage analysis of the phosphoproteome has largely been achieved by combining pre-fractionation methods with multiple phosphopeptide enrichment approaches, at some cost in LC-MS/MS measurement time and increased sample loss. Key points for the future will be to further increase the selectivity and the recovery of enrichment methods to achieve higher sensitivity and efficiency in LC-MS/MS analysis in order to detect protein phosphorylation comprehensively, including low-abundance proteins. This is expected to lead to a more detailed understanding of the mechanisms and interactions of phosphorylation-mediated regulatory pathways in biological systems.

Large-scale identification of phosphorylation sites for profiling protein kinase selectivity.

Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of in vitro kinase reaction followed by LC-MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO2-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC-MS/MS. As a result, 3585, 4347, and 1778 in vitro phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites.

Temporal profiling of lapatinib-suppressed phosphorylation signals in EGFR/HER2 pathways.

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 µm lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049-1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.

Nicotinamide mononucleotide (NMN) alleviates the poly(I:C)-induced inflammatory response in human primary cell cultures.

NMN is the direct precursor of nicotinamide adenine dinucleotide (NAD+) and is considered as a key factor for increasing NAD+ levels and mitochondrial activity in cells. In this study, based on transcriptome analysis, we showed that NMN alleviates the poly(I:C)-induced inflammatory response in cultures of two types of human primary cells, human pulmonary microvascular endothelial cells (HPMECs) and human coronary artery endothelial cells (HCAECs). Major inflammatory mediators, including IL6 and PARP family members, were grouped into coexpressed gene modules and significantly downregulated under NMN exposure in poly(I:C)-activated conditions in both cell types. The Bayesian network analysis of module hub genes predicted common genes, including eukaryotic translation initiation factor 4B (EIF4B), and distinct genes, such as platelet-derived growth factor binding molecules, in HCAECs, which potentially regulate the identified inflammation modules. These results suggest a robust regulatory mechanism by which NMN alleviates inflammatory pathway activation, which may open up the possibility of a new role for NMN replenishment in the treatment of chronic or acute inflammation.

Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data.

BACKGROUND: Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches. RESULTS: We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC) method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF). We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm) tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR) signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled. CONCLUSIONS: The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics-based network, our method will be a complementary strategy to explore new components of protein signaling pathways in combination with previous methods (including software) of predicting direct kinase-substrate relationships.

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Large-scale Discovery of Substrates of the Human Kinome.

Kinase networks are important for cellular signal transduction. Despite tremendous efforts to uncover these signaling pathways, huge numbers of uncharacterized phosphosites still remain in the human proteome. Because of the transient nature of kinase-substrate interactions in vivo, it is almost impossible to identify direct substrates. Here, we present a strategy for the rapid, accurate and high-throughput discovery of in vitro kinase substrates using quantitative proteomics. Using 385 purified kinases (354 wild-type protein kinases, 21 mutants and 10 lipid kinases), we identified a total of 175,574 potential direct kinase substrates. In addition, we identified novel kinase groups, such as one group containing 30 threonine-directed kinases and another containing 15 serine/threonine/tyrosine kinases. Surprisingly, we observed that the diversity of substrates for tyrosine kinases was much higher than that for serine-threonine kinases.