The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria.     

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.     

Using phosphoproteomics to reveal signalling dynamics in plants

To ensure appropriate responses to stimuli, organisms have evolved signalling networks that rely on post-translational modifications of their components. Among these, protein phosphorylation has a prominent role and much research in plants has focused on protein kinases and phosphatases, which, respectively, catalyse phosphorylation and dephosphorylation of specific substrates. Technical limitations, however, have hampered the identification of these substrates. As reviewed here, novel mass spectrometry-based techniques have enabled the large-scale mapping of in vivo phosphorylation sites. Alternatively, methods based on peptide and protein microarrays have revealed protein kinase activities in cell extracts, in addition to kinase substrates. A combined phosphoproteomic approach of mass spectrometry and microarray technology could enhance the construction of dynamic plant signalling networks that underlie plant biology.     

Protein-tyrosine phosphorylation in Bacillus subtilis

In recent years bacterial protein-tyrosine kinases have been found to phosphorylate a growing number of protein substrates, including RNA polymerase sigma factors, UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. The activity of these protein substrates was affected by tyrosine phosphorylation, indicating that this post-translational modification could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this field was done in Bacillus subtilis, and we here present the current state of knowledge on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specificity and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology.     

MAP kinase phosphatases

Mitogen-activated protein MAP kinases are key signal-transducing enzymes that are activated by a wide range of extracellular stimuli. They are responsible for the induction of a number of cellular responses, such as changes in gene expression, proliferation, differentiation, cell cycle arrest and apoptosis. Although regulation of MAP kinases by a phosphorylation cascade has long been recognized as significant, their inactivation through the action of specific phosphatases has been less studied. An emerging family of structurally distinct dual-specificity serine, threonine and tyrosine phosphatases that act on MAP kinases consists of ten members in mammals, and members have been found in animals, plants and yeast. Three subgroups have been identified that differ in exon structure, sequence and substrate specificity.     

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts.

BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of extracellular signal regulated (Erk) MAP kinases. CONCLUSIONS: These observations demonstrate that RPTPalpha functions as a physiological upstream activator of Src-family kinases in fibroblasts and establish this tyrosine phosphatase as a newly identified regulator of integrin signalling.     

蛋白组氨酸磷酸酶研究进展

主要概括磷酸酶的种类,原核细胞磷酸组氨酸生物功能及调控,哺乳动物组氨酸残基磷酸化、去磷酸化,以及组氨酸磷酸酶及其底物的最新研究进展. 信号转导在生长发育及细胞功能中起极其重要的作用. 无论在原核还是真核细胞,蛋白质磷酸化是细胞内信号转导的关键机制. 研究最多的可逆的真核蛋白磷酸化,主要发生在含有羟基的丝氨酸、苏氨酸和酪氨酸残基上. 不同的激酶和磷酸酶受不同机制的调节,而调节过程中出现的差异是人类很多疾病的潜在基础. 与大量有关羟基磷酸化氨基酸的报道相比,有关氨基磷酸化氨基酸的报道甚少. 据估计,自然界中存在的磷酸组氨酸比磷酸酪氨酸多10 ~ 100倍,但不如磷酸丝氨酸丰富. 虽然对脊椎动物蛋白质中存在磷酸组氨酸的认识可以追溯到20世纪60年代初, 但由于研究手段的限制,至今对脊椎动物蛋白组氨酸激酶及组氨酸磷酸酶的结构及功能知之甚少. 但是,近几年的研究有突破性的发现,克隆和重组表达哺乳动物组氨酸磷酸酶为研究氨基磷酸化氨基酸的生物功能翻开新的一章.     

Multifaceted roles of Arabidopsis PP6 phosphatase in regulating cellular signaling and plant development

Reversible protein phosphorylation catalyzed by kinases and phosphatases is a major form of posttranslational regulation that plays a central role in regulating many signaling pathways. While large families of both protein kinases and protein phosphatases have been identified in plants, kinases outnumber phosphatases. This raises the question of how a relatively limited number of protein phosphatases can maintain protein phosphorylation homeostasis in a cell. Recent studies have shown that Arabidopsis FyPP1 (Phytochrome-associated serine/threonine protein phosphatase 1) and FyPP3 encode the catalytic subunits of protein phosphatase 6 (PP6), and that they directly binds to the A subunits of protein phosphatase 2A (PP2AA proteins), and SAL (SAPS domain-like) proteins to form the heterotrimeric PP6 holoenzyme complex. Emerging evidence is suggesting that PP6, acts in opposition with multiple classes of kinases, to regulate the phosphorylation status of diverse substrates and subsequently numerous developmental processes and responses to environmental stimuli.     

The Tyrosine Kinase Csk Dimerizes through Its SH3 Domain

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine. The SH3 domain of Csk recruits the tyrosine phosphatase PEP, allowing for the coordinated inhibition of Src family kinase activity. We have discovered that Csk forms homodimers through interactions mediated by the SH3 domain in a manner that buries the recognition surface for SH3 ligands. The formation of this dimer would therefore block the recruitment of tyrosine phosphatases and may have important implications for the regulation of Src kinase activity.     

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.     

Prediction of substrate sites for protein phosphatases 1B,SHP-1, and SHP-2 based on sequence features

Tyrosine phosphorylation plays crucial roles in numerous physiological processes. The level of phosphorylation state depends on the combined action of protein tyrosine kinases and protein tyrosine phosphatases. Detection of possible phosphorylation and dephosphorylation sites can provide useful information to the functional studies of relevant proteins. Several studies have focused on the identification of protein tyrosine kinase substrates. However, compared with protein tyrosine kinases, the prediction of protein tyrosine phosphatase substrates involved in the balance of protein phosphorylation level falls behind. This paper described a method that utilized the k-nearest neighbor algorithm to identity the substrate sites of three protein tyrosine phosphatases based on the sequence features of manually collected dephosphorylation sites. In the performance evaluation, both sensitivities and specificities could reach above 75 % for all three protein tyrosine phosphatases. Finally, the method was applied on a set of known tyrosine phosphorylation sites to search for candidate substrates.     

Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.     

Essential functions of protein tyrosine phosphatases PTP2 and PTP3 and RIM11 tyrosine phosphorylation in Saccharomyces cerevisiae meiosis and sporulation

Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3 results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Deltaptp3Delta homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation     

Mammalian histidine kinases

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

高等植物中的蛋白磷酸酶与信号传递途径

蛋白激酶与蛋白磷酸酶在细胞信号传递中起着重要作用。有关高等植物中蛋白激酶的研究工作已经较多,但关于蛋白磷酸酶的研究在以前却未受到足够的重视。本文主要介绍最近有关蛋白磷酸酶在高等植物的信号传递中有重要作用的研究工作。这些与蛋白磷酸酶有关的信号传递途径包括气孔运动调节与脱落酸的信号转导、植物对病原及逆境的响应以及植物发育的调控。这些研究工作清楚地证明,蛋白磷酸酶的功能不仅表现为蛋白激酶功能的逆向平衡机制,而且在许多信号传递过程中蛋白磷酸酶起着主导作用。