Mitogenesis and protein synthesis: A role for ribosomal protein S6 phosphorylation?

It has been known for 20 years that the ribosomal protein S6 is rapidly phosphorylated when cells are stimulated to grow or divide. Furthermore, numerous studies have documented that there is a strong correlation between increases in S6 phosphorylation and protein synthesis, leading to the idea that S6 phosphorylation is involved in up-regulating translation. In an attempt to define a mechanism by which S6 phosphorylation exerts translational control, other studies have focused on characterizing the sites of phosphorylation of this protein and its location within the ribosome. Recent data show that S6 is a protein which may have diverse cellular functions and is essential for normal development, and that it may be involved in the translational regulation of a specific class of messages.     

Computational Analysis of the Predicted Evolutionary Conservation of Human Phosphorylation Sites

Protein kinase-mediated phosphorylation is among the most important post-translational modifications. However, few phosphorylation sites have been experimentally identified for most species, making it difficult to determine the degree to which phosphorylation sites are conserved. The goal of this study was to use computational methods to characterize the conservation of human phosphorylation sites in a wide variety of eukaryotes. Using experimentally-determined human sites as input, homologous phosphorylation sites were predicted in all 432 eukaryotes for which complete proteomes were available. For each pair of species, we calculated phosphorylation site conservation as the number of phosphorylation sites found in both species divided by the number found in at least one of the two species. A clustering of the species based on this conservation measure was concordant with phylogenies based on traditional genomic measures. For a subset of the 432 species, phosphorylation site conservation was compared to conservation of both protein kinases and proteins in general. Protein kinases exhibited the highest degree of conservation, while general proteins were less conserved and phosphorylation sites were least conserved. Although preliminary, these data tentatively suggest that variation in phosphorylation sites may play a larger role in explaining phenotypic differences among organisms than differences in the complements of protein kinases or general proteins.     

Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.     

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.     

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.     

Phosphoproteomics toolbox: computational biology, protein chemistry and mass spectrometry

Protein phosphorylation is important for regulation of most biological functions and up to 50% of all proteins are thought to be modified by protein kinases. Increased knowledge about potential phosphorylation of a protein may increase our understanding of the molecular processes in which it takes part. Despite the importance of protein phosphorylation, identification of phosphoproteins and localization of phosphorylation sites is still a major challenge in proteomics. However, high-throughput methods for identification of phosphoproteins are being developed, in particular within the fields of bioinformatics and mass spectrometry. In this review, we present a toolbox of current technology applied in phosphoproteomics including computational prediction, chemical approaches and mass spectrometry-based analysis, and propose an integrated strategy for experimental phosphoproteomics.     

A differential phosphoproteomic analysis of retinoic acid-treated P19 cells

External stimuli trigger internal signaling events within a cell that may represent either a temporary or permanent shift in the phosphorylation state of its proteome. Numerous reports have elucidated phosphorylation sites from a variety of biological samples and more recent studies have monitored the temporal dynamics of protein phosphorylation as a given system is perturbed. Understanding which proteins are phosphorylated as well as when they are phosphorylated may indicate novel functional roles within a system and allow new therapeutic avenues to be explored. To elucidate the dynamics of protein phosphorylation within differentiating murine P19 embryonal carcinoma cells, we induced P19 cells to differentiate using all-trans-retinoic acid and developed a strategy that combines isotopically labeled methyl esterification, immobilized metal affinity chromatography, mass spectrometric analysis, and a rigorous and unique data evaluation approach. We present the largest differential phosphoproteomic analysis using isotopically labeled methyl esterification to date, identifying a total of 472 phosphorylation sites on 151 proteins; 56 of these proteins had altered abundances following treatment with retinoic acid and approximately one-third of these have been previously associated with cellular differentiation. A series of bioinformatic tools were used to extract information from the data and explore the implications of our findings. This study represents the first global gel-free analysis that elucidates protein phosphorylation dynamics during cellular differentiation.     

A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.     

Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

Effects of phosphorylation of P-glycoprotein on multidrug resistance

Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype. Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter. Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to their effects on the state of phosphorylation of P-glycoprotein. The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested. Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein. Mutational analyses have been initiated to confirm this finding. Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

The role of the VQIVYK peptide in tau protein phosphorylation

Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306–311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306–311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau–tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.     

Prediction of functional phosphorylation sites by incorporating evolutionary information

Protein phosphorylation is a ubiquitous protein post-translational modification, which plays an important role in cellular signaling systems underlying various physiological and pathological processes. Current in silico methods mainly focused on the prediction of phosphorylation sites, but rare methods considered whether a phosphorylation site is functional or not. Since functional phosphorylation sites are more valuable for further experimental research and a proportion of phosphorylation sites have no direct functional effects, the prediction of functional phosphorylation sites is quite necessary for this research area. Previous studies have shown that functional phosphorylation sites are more conserved than non-functional phosphorylation sites in evolution. Thus, in our method, we developed a web server by integrating existing phosphorylation site prediction methods, as well as both absolute and relative evolutionary conservation scores to predict the most likely functional phosphorylation sites. Using our method, we predicted the most likely functional sites of the human, rat and mouse proteomes and built a database for the predicted sites. By the analysis of overall prediction results, we demonstrated that protein phosphorylation plays an important role in all the enriched KEGG pathways. By the analysis of protein-specific prediction results, we demonstrated the usefulness of our method for individual protein studies. Our method would help to characterize the most likely functional phosphorylation sites for further studies in this research area.     

Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates

Protein phosphorylation is a highly conserved mechanism for regulating protein function, being found in all prokaryotes and eukaryotes examined. Phosphorylation can alter protein activity or subcellular localization, target proteins for degradation and effect dynamic changes in protein complexes. In many cases, different kinases may be involved in each of these processes for a single protein, allowing a large degree of combinatorial regulation at the post-translational level. Therefore, knowing which kinases are activated during a response and which proteins are substrates is integral to understanding the mechanistic regulation of a wide range of biological processes. In this paper, I will describe methods for monitoring kinase activity, investigating kinase-substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed.     

Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methods

Protein phosphorylation activates or deactivates many other proteins especially protein enzymes, and plays a significant role in a wide range of cellular processes. Recent advances in phosphopeptide enrichment procedures and mass spectrometry-based peptide sequencing techniques have enabled us to identify large number of protein phosphorylation sites. In this study, we combined three different HPLC techniques in fractionating enriched phosphopeptides before RPLC-MS/MS analysis, and found that although between 4000-5000 unique phosphopeptides could be identified following any of the HPLC fraction method, different HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides and 3260 phosphoproteins covering 9463 unique phosphorylation sites, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motifs were also discovered from our study.     

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.     

Prediction of PK-specific phosphorylation site based on information entropy

Phosphorylation is a crucial way to control the activity of proteins in many eukaryotic organisms in vivo. Experimental methods to determine phosphorylation sites in substrates are usually restricted by the in vitro condition of enzymes and very intensive in time and labor. Although some in silico methods and web servers have been introduced for automatic detection of phosphorylation sites, sophisticated methods are still in urgent demand to further improve prediction performances. Protein primary sequences can help predict phosphorylation sites catalyzed by different protein kinase and most computational approaches use a short local peptide to make prediction. However, the useful information may be lost if only the conservative residues that are not close to the phosphorylation site are considered in prediction, which would hamper the prediction results. A novel prediction method named IEPP (Information-Entropy based Phosphorylation Prediction) is presented in this paper for automatic detection of potential phosphorylation sites. In prediction, the sites around the phosphorylation sites are selected or excluded by their entropy values. The algorithm was compared with other methods such as GSP and PPSP on the ABL, MAPK and PKA PK families. The superior prediction accuracies were obtained in various measurements such as sensitivity (Sn) and specificity (Sp). Furthermore, compared with some online prediction web servers on the new discovered phosphorylation sites, IEPP also yielded the best performance. IEPP is another useful computational resource for identification of PK-specific phosphorylation sites and it also has the advantages of simpleness, efficiency and convenience.