Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established

The first clearly established example of Ser/Thr/Tyr phosphorylation of a bacterial protein was isocitrate dehydrogenase. In 1979, 25 years after the discovery of protein phosphorylation in eukaryotes, this enzyme was reported to become phosphorylated on a serine residue. In subsequent years, numerous other bacterial proteins phosphorylated on Ser, Thr or Tyr were discovered and the corresponding protein kinases and P-protein phosphatases were identified. These protein modifications regulate all kinds of physiological processes. Ser/Thr/Tyr phosphorylation in bacteria therefore seems to play a similar important role as in eukaryotes. Surprisingly, many bacterial protein kinases do not exhibit any similarity to eukaryotic protein kinases, but rather resemble nucleotide-binding proteins or kinases phosphorylating diverse low-molecular-weight substrates.     

The extensive and functionally uncharacterized mitochondrial phosphoproteome

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis.     

Protein tyrosine kinases and phosphatases in the nervous system.

Evidence in the past year has provided support for a prominent role of tyrosine phosphorylation in the regulation of neuronal function. The discovery that many novel forms of protein tyrosine kinases and phosphatases are expressed in the brain has revealed that the regulation of tyrosine phosphorylation is highly complex. The recent identification of substrate proteins in the brain for the protein tyrosine kinases and phosphatases has begun to clarify the functional role of tyrosine phosphorylation in the development and modulation of the nervous system.     

On the importance of protein phosphorylation in cell cycle control

This chapter is written as a contribution to a volume commemorating the work of Krebs and Fischer that led to awarding of the Nobel Prize in 1992. This award was made because of their fundamental discovery in the mid-1950s that protein phosphorylation was the underlying mechanism that accounted for the reversible modification of activity of glycogen phosphorylase in mammalian skeletal muscle. Although it could not be anticipated at the time that phosphorylation would turn out to be such a ubiquitous regulator of cellular functions, it is now evident that phosphorylation controls virtually every important reaction in cells and provides the basis for understanding how integrated cellular behavior is regulated by both extracellular signals and internal control mechanisms. This chapter relates the historical development in biochemical terms of protein phosphorylation as a regulator of the cell cycle in Xenopus oocytes and eggs.     

Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology

Tyrosine phosphorylation is a key device in numerous cellular functions in eukaryotes, but in bacteria this protein modification was largely ignored until the mid-1990s. The first conclusive evidence of bacterial tyrosine phosphorylation came only a decade ago. Since then, several tyrosine kinases exhibiting unexpected features have been identified in a variety of bacteria. These enzymes use homologues of Walker motifs of nucleotide-binding proteins for their catalytic mechanism, thus defining an idiosyncratic type of bacterial tyrosine kinases. Recently, bacterial tyrosine kinases have been found to phosphorylate an increasing list of endogenous protein substrates. This discovery contributes to the emerging picture that bacterial tyrosine phosphorylation is an important regulatory arsenal of bacterial physiology in addition to the classical serine/threonine kinases, and the 'two-component' and phosphotransferase systems.     

Pheromone signaling pathways in yeast.

The discovery that the transducing G protein of the Saccharomyces cerevisiae mating pheromone response figures centrally in signal adaptation was the focus of considerable excitement in the past year. Not only does activated G alpha in this system stimulate an adaptive signal but G beta undergoes a desensitizing phosphorylation in response to pheromone signaling.     

Phosphoprotein analysis: from proteins to proteomes

Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation.     

Activation of guanylate cyclase by natriuretic peptides in mouse pituitary AtT20 cells is influenced by phosphorylation of ANP

The discovery of free and membrane-bound ectokinases raises the question whether phosphorylation is another mechanism to modulate the action of distinct neuropeptides. Atrial-natriuretic-peptide (ANP) which is widespread found in the central nervous system (CNS) and involved in the modulation of stress reactions and emotional states like anxiety contains a recognition-motif for cAMP-dependent protein kinase A. We investigated the effect of phosphorylation of ANP and C-type natriuretic peptide (CNP), a related peptide without phosphorylation site, on their ability to activate their receptors in mouse pituitary AtT20 cells by measuring the formation of cyclic guanosinmonophosphate (cGMP). Phosphorylation with protein kinase A inactivated ANP. Coincubation experiments adding adenosintriphosphate (ATP), ATP-analogues or inhibitors of protein kinases to the medium pointed to the presence of an intrinsic protein kinase A like ectokinase-activity on AtT20 cells. The activity of CNP was unaffected in these experiments. Phosphorylation by ectokinases may be a physiological mechanism to regulate the biological activity of ANP in different tissues, such as pituitary and CNS.     

Protein phosphorylation of nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor (nAcChR) is a ligand-gated ion channel found in the postsynaptic membranes of electric organs, at the neuromuscular junction, and at nicotinic cholinergic synapses of the mammalian central and peripheral nervous system. The nAcChR from Torpedo electric organ and mammalian muscle is the most well-characterized neurotransmitter receptor in biology. It has been shown to be comprised of five homologous (two identicle) protein subunits (alpha 2 beta gamma delta) that form both the ion channel and the neurotransmitter receptor. The nAcChR has been purified and reconstituted into lipid vesicles with retention of ion channel function and the primary structure of all four protein subunits has been determined. Protein phosphorylation is a major posttranslational modification known to regulate protein function. The Torpedo nAcChR was first shown to be regulated by phosphorylation by the discovery that postsynaptic membranes contain protein kinases that phosphorylate the nAcChR. Phosphorylation of the nAcChR has since been shown to be regulated by the cAMP-dependent protein kinase, protein kinase C, and a tyrosine-specific protein kinase. Phosphorylation of the nAcChR by cAMP-dependent protein kinase has been shown to increase the rate of nAcChR desensitization, the process by which the nAcChR becomes inactivated in the continued presence of agonist. In cultured muscle cells, phosphorylation of the nAcChR has been shown to be regulated by cAMP-dependent protein kinase, a Ca2+-sensitive protein kinase, and a tyrosine-specific protein kinase. Stimulation of the cAMP-dependent protein kinase in muscle also increases the rate of nAcChR desensitization and correlates well with the increase in nAcChR phosphorylation. The AcChR represents a model system for how receptors and ion channels are regulated by second messengers and protein phosphorylation.     

Reversible phosphorylation of histidine residues in vertebrate proteins

Knowledge on kinases and phosphatases acting on serine, threonine and tyrosine residues of vertebrate proteins is huge. These enzymes are still under intensive investigation at present. This is in sharp contrast to what is known about kinases and phosphatases acting on histidine, arginine, lysine and aspartate residues in vertebrate proteins. It also is in contrast to extensive studies of histidine/aspartate phosphorylation in prokaryotes. This minireview briefly summarizes what we have learned about the reversible phosphorylation of histidine residues in mammals. It is described how the field developed during 40 years of science. The article especially highlights the discovery of the first protein histidine phosphatase from vertebrates. Having identified and characterized a protein histidine phosphatase provides at least one desperately required tool to handle and study phosphorylation and dephosphorylation of histidine residues in vertebrates in more detail. Recent evidence even suggests an involvement of histidine phosphorylation in signal transduction.     

Will the ubiquitin system furnish as many drug targets as protein kinases?

Protein phosphorylation and protein ubiquitination regulate most aspects of cell life, and defects in these control mechanisms cause cancer and many other diseases. In the past decade, protein kinases have become one of the most important classes of drug targets for the pharmaceutical industry. In contrast, drug discovery programs that target components of the ubiquitin system have lagged behind. In this Perspective, we discuss the reasons for the delay in this pipeline, the drugs targeting the ubiquitin system that have been developed, and new approaches that may popularize this area of drug discovery in the future.     

Pathway illuminated: visualizing protein kinase C signaling

Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.     

Cellular regulation by protein phosphorylation

A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca²⁺ and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.     

植物蛋白激酶研究进展

近年来,在分子生物学技术不断完善和在酵母与动物蛋白激酶研究的基础上,植物蛋白激酶的研究已取得了很大的进展。就近十年来国内外学者对植物蛋白激酶的发现,家族分类,磷酸化过程及其生理功能等方面的研究进行综述。最后分析了存在的问题并对今后的研究提出了展望。     

Specific phosphorylation of exogenous protein and peptide substrates by the human cytomegalovirus UL97 protein kinase. Importance of the P+5 position

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified (32)P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.     

Surface Phosphorylation by Ecto-Protein Kinase C in Brain Neurons: A Target for Alzheimer's β-Amyloid Peptides

Abstract: The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ectoprotein kinase (ecto-PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC-pseudosubstrate 19–36; K252b) and antibodies (anti-PKC catalytic region M.Ab.1.9, antibodies to the carboxy-terminus of eight PKC isozymes) revealed several types of ecto-PK activity, among them ecto-PKs with catalytic specificity of the PKC isozymes ζ and δ. The activity of the neuronal ecto-PKC is constitutive and not stimulated by phorbol esters. The phosphorylation of a 12K/13K surface protein duplex by ecto-PKC-δ was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides β1–40 and β25–35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto-PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose-response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, β-amyloid peptide 1–28 did not inhibit this surface phosphorylation. The discovery that ecto-PKC-mediated protein phosphorylation serves as a target for β-amyloid peptides at the very site they operate, i.e., at the neuronal cell surface, opens a new research direction in the investigation of molecular events that play a role in the etiology of developmental disabilities and neurodegenerative disorders.     

Protein kinase CK2 phosphorylates BAD at threonine-117

Reversible phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated phosphorylation events on BAD.     

Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (<1% of total protein phosphorylation), only a few tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca(2+) homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states.     

Quantitative phosphoproteomic analysis of signaling network dynamics

Protein phosphorylation mediated cellular signaling is a highly regulated, dynamic process that controls many aspects of cellular biology. Over the past few years many methods have been developed to quantify temporal dynamics of protein phosphorylation, including mass spectrometry, which can be applied in both an unbiased, discovery mode and in a targeted mode to monitor specific phosphorylation sites. Other methods, such as kinase activity assays and antibody microarrays, have been applied to quantify central nodes in the signaling network, yielding intriguing biological insights. This review provides a concise overview of the latest advances in the quantitative analysis of signaling dynamics including a brief commentary on the future of the field.     

Protein kinase substrate identification on functional protein arrays

Background  

Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms.