Identification of the rapamycin-sensitive phosphorylation sites within the Ser/Thr-rich domain of the yeast Npr1 protein kinase

The Saccharomyces cerevisae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a fungus-specific family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the TOR (target of rapamycin) signalling pathway. Inhibition of the TOR proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. To identify the rapamycin-sensitive phosphorylation sites in yeast Npr1, glutathione-S-transferase (GST)-tagged Npr1 was isolated from untreated or rapamycin-treated cells, and analyzed by mass spectrometry. Here, we report for the first time 22 phosphorylation sites that are clustered in two regions of the N-terminal serine-rich domain. All phosphorylation sites, except two, were found to be rapamycin-sensitive. Some phosphorylation sites are contained in motifs that closely resemble those in mammalian S6K (serines followed by a tyrosine or a phenylalanine) and 4E-BP1 (serines followed by a proline). Other sites, such as serines followed by Ala, Asn, Gln, His, Ile, Leu, or Val, appear to define new motifs. Thus, TOR controls an unusually broad array of phosphorylation sites in Npr1. In addition to phosphorylation by upstream kinases, Npr1 undergoes autophosphorylation that was mapped to three distinct serines in the N-terminal domain of which Ser257 appears to be the main autophosphorylation site. Site-directed mutagenesis confirmed the mass spectral assignments of the autophosphorylation sites and shows that Ser257 is specifically involved in forming an in vitro substrate-binding site.     

Polyprenyl-hydroquinones and -furans from three marine sponges inhibit the cell cycle regulating phosphatase CDC25A

The CDC25 phosphatases regulate the cell division cycle by controlling the activity of cyclin-dependent kinases. While screening for inhibitors of phosphatases among natural products we repeatedly found that some polyprenyl-hydroquinones and polyprenyl-furans (furanoterpenoids) (furospongins, furospinosulins) were potent CDC25 phosphatase inhibitors. These compounds were extracted, isolated and identified independently from three sponge species (Spongia officinalis, Ircinia spinulosa, Ircinia muscarum), collected at different locations in the Mediterranean Sea. The compounds were inactive on the Ser/Thr phosphatase PP2C-alpha and on three kinases (CDK1, CDK5, GSK-3), suggesting that some potent and selective CDC25 phosphatase might be designed from these initial structures.     

Design of a protein kinase-inducible domain

Protein phosphorylation is a critical regulatory strategy. New tools are necessary which may be used to interrogate and are responsive to the activities of protein kinases and phosphatases. We have used protein design to develop a protein motif, termed a protein kinase-inducible domain, whose structure is dependent on its phosphorylation state. Based on an EF hand calcium-binding loop, the key design element is the replacement of a structurally critical Glu residue, which binds metal in a bidentate manner, with a serine residue, which is expected to bind metal tightly when phosphorylated but poorly when not phosphorylated. The design comprises an EF hand consensus sequence, a tryptophan at residue 7 to sensitize lanthanide luminescence, and the recognition sequence of a serine/threonine kinase. Designed peptides, which contain minimal substrate recognition motifs of the protein kinases PKA, PKC, or the MAP kinase Erk, form complexes with Tb3+ when phosphorylated, showing strong Tb3+ luminescence emission at 544 nm, but show weak luminescence when not phosphorylated. The change in fluorescence on phosphorylation is comparable to or greater than that observed in described kinase sensors. Site-specific lanthanide binding was confirmed by NMR with diamagnetic and paramagnetic metals. The kinase-inducible domain peptides comprise an expressible sequence, potentially enabling their use as genetically encoded tags of protein kinase activity. The motif is general and potentially applicable to the majority of serine/threonine kinases.     

Recognition of multiple substrate motifs by the c-ABL protein tyrosine kinase

Using a combinatorial peptide library that is based on the one-bead one-peptide approach we identified 14 peptide substrates for the c-ABL protein tyrosine kinase, which define three distinct consensus sequence groups. This is distinct from many serine/threonine kinases, which often phosphorylate only one major consensus sequence. The three consensus sequences accurately predict phosphorylation sites in cellular ABL substrates proven to play a role in cell signaling. Our data suggest that protein tyrosine kinases have evolved to recognize multiple substrate motifs.     

Structural analysis of the protein phosphatase 1 docking motif: molecular description of binding specificities identifies interacting proteins

The interplay between kinases and phosphatases represents a fundamental regulatory mechanism in biological systems. Being less numerous than kinases, phosphatases increase their diversity by the acquisition of a variety of binding partners, thereby forming a large number of holoenzymes. Proteins interacting with protein phosphatase 1 (PP1) often bind via a so-called docking motif to regulate its enzymatic activity, substrate specificity, and subcellular localization. Here, we systematically determined structural elements that mediate the binding specificity of PP1 interacting proteins, and propose a refined consensus sequence for high-affinity PP1 ligands. Applying this pattern to database searches, we predicted and experimentally confirmed several previously unknown PP1 interactors. Thus, the suggested PP1 docking motif enables a highly specific prediction of PP1 binding partners, thereby facilitating the genome-wide identification of PP1 interactors.     

Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phosphospecific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r² = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation.     

4-Methylpseudoproline derived from α-methylserine - synthesis and conformational studies

This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6a) and Fmoc-Ala-(S)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6b). Additionally, the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is additionally influenced by the presence of two symmetrically independent molecules in an asymmetric unit. Both molecules adopt a conformation which resembles β-turn type II, stabilized by hydrogen bonding. The conformational preferences and prolyl cis-trans isomerization of Ac-(αMe)Ser(Ψ(H,H)Pro)-NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the amide bonds, with lower puckering in the transition state of the cis-trans isomerization.     

UVA, UVB and UVC induce differential response signaling pathways converged on the eIF2α phosphorylation

It is clear that solar UV irradiation is a crucial environmental factor resulting in skin diseases partially through activation of cell signaling toward altered gene expression and reprogrammed protein translation. Such a key translational control mechanism is executed by the eukaryotic initiation factor 2α subunit (eIF2α) and the downstream events provoked by phosphorylation of eIF2α at Ser(51) are clearly understood, but the upstream signaling mechanisms on the eIF2α-Ser(51) phosphorylation responses to different types of UV irradiations, namely UVA, UVB and UVC, are still not well elucidated. Herein, our evidence reveals that UVA, UVB and UVC all induce a dose- and time-dependent phosphorylation of eIF2α-Ser(51) through distinct signaling mechanisms. UVA-induced eIF2α phosphorylation occurs through MAPKs, including ERKs, JNKs and p38 kinase, and phosphatidylinositol (PI)-3 kinase. By contrast, UVB-induced eIF2α phosphorylation is through JNKs and p38 kinase, but not ERKs or PI-3 kinase, whereas UVC-stimulated response to eIF2α phosphorylation is via JNKs alone. Furthermore, we have revealed that ATM is involved in induction of the intracellular responses to UVA and UVB, rather than UVC. These findings demonstrate that wavelength-specific UV irradiations activate differential response signaling pathways converged on the eIF2α phosphorylation. Importantly, we also show evidence that a direct eIF2α kinase PKR is activated though phosphorylation by either RSK1 or MSK1, two downstream kinases of MAPKs/PI-3 kinase-mediated signaling pathways.     

Protein posttranslational modifications: the chemistry of proteome diversifications

The diversity of distinct covalent forms of proteins (the proteome) greatly exceeds the number of proteins predicted by DNA coding capacities owing to directed posttranslational modifications. Enzymes dedicated to such protein modifications include 500 human protein kinases, 150 protein phosphatases, and 500 proteases. The major types of protein covalent modifications, such as phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation, can be classified according to the type of amino acid side chain modified, the category of the modifying enzyme, and the extent of reversibility. Chemical events such as protein splicing, green fluorescent protein maturation, and proteasome autoactivations also represent posttranslational modifications. An understanding of the scope and pattern of the many posttranslational modifications in eukaryotic cells provides insight into the function and dynamics of proteome compositions.     

Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.     

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro

Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD). Endothelial cells play a pivotal role in CVD initiation and development; however, there have been limited investigations of the specific signaling cascades and protein phosphorylations activated by cigarette smoke in endothelial cells. The purpose of this research was to better understand the differential protein phosphorylation in endothelial cells stimulated with extracts of cigarette smoke total particulate matter (CS-TPM) in vitro. Human microvascular endothelial cells were exposed in vitro to CS-TPM at concentrations that were shown to cause endothelial cell dysfunction. The phosphorylated proteins were isolated using phosphoprotein-specific chromatography, followed by enzymatic digestion and nano-flow capillary liquid chromatography (ncap-LC) coupled to high resolution mass spectrometry. This study putatively identified 94 proteins in human microvascular endothelial cells that were differentially bound to a phosphoprotein-specific chromatography column following exposure to CS-TPM suggesting differential phosphorylation. Pathway analysis has also been conducted and confirmations of several observations have been made using immunoaffinity-based techniques (e.g., Western blotting). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pharmacoinformatics and molecular dynamic simulation studies to identify potential small-molecule inhibitors of WNK-SPAK/OSR1 signaling that mimic the RFQV motifs of WNK kinases

The WNK-SPAK/OSR1 signaling is a complex of serine and threonine protein kinases that involves in the regulation of human blood pressure. The WNK kinases phosphorylate and activate SPAK and OSR1 kinases through the interaction of RFQV motifs of WNK kinases with the C-terminal domains of SPAK and OSR1. Upon phosphorylation, SPAK and OSR1 phosphorylate key ion co-transporters such as Na⁺-[K⁺]-2Cl⁻ (NKCC_1-2) and K⁺-Cl⁻ (KCC_1-4), which are essential for electrolytes balance and blood pressure regulation. Targeting the binding site of the RFQV motifs of WNK kinases on the C-terminal domain (CTD) of SPAK and OSR1 has emerged as a valuable approach to inhibit the WNK-SPAK/OSR1 signaling pathway. Herein, an effort has been intended to pinpoint non-peptidic small-molecules that could disrupt the binding of SPAK/OSR1 to WNK kinases, hence, inhibit the SPAK and OSR1 phosphorylation and activation by WNK kinases through pharmacoinformatics and molecular dynamic simulation methodologies. A sequential structure-based virtual screening of a focus protein-protein interaction chemical library composed of 11,870 compounds lead to the identification of three compounds having good lead-compound properties with respect to their predicted inhibitory constants, pharmacophore fit scores, binding affinities, ADME-T parameters, drug-likeness properties and ligand efficiency metrics. The mechanism of interaction and binding stability of these compounds to OSR1-CTD were confirmed using molecular docking and dynamic simulation studies. Hence, the identified compounds may have therapeutic potential as novel antihypertensive agents subjected to experimental validation.     

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two‐dimensional gel electrophoresis

2‐DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2‐D gels. Of the 588 matched proteins separated on 2‐D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein‐specific staining was corroborated by the identification of 16 phosphoproteins by nano‐LC MS/MS and phosphotyrosine kinase activity assay.     

一种位点注释的蛋白质数据库用于磷酸化肽段的鉴定

磷酸化修饰的分析一直是蛋白质组学研究的热点之一.在鸟枪法的蛋白质组学研究中,通过在数据库检索中设定磷酸化为可变修饰可以直接鉴定磷酸化修饰的位点.但是翻译后修饰的引入会增加数据检索空间,造成鉴定灵敏度的降低.为了解决这一问题,我们构建了一种位点注释的数据库,这种数据库包含蛋白质的磷酸化位点信息,并开发了一种新的数据库检索策略用于磷酸化肽段的可靠鉴定.用不同类型的数据作为分析对象,通过Mascot检索软件对这种新的数据库检索策略进行了考察,证明了这种方法在保证鉴定结果可靠性的前提下提高了磷酸化肽段鉴定的灵敏度.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

Transmembrane Signaling of T Lymphocytes by Ligand-Induced Receptor Complex Assembly

T lymphocytes (T cells) are the central cell type initiating all immune responses. They are able to recognize other cells in the body that have been invaded by foreign living or nonliving matter. In such cells, foreign peptides generated by intracellular breakdown are complexed with molecules of the major histocompatibility complex (MHC) specially designed for peptide binding. Peptide-loaded MHC molecules appear on the surface of these cells and alert the immune system. The molecular complex which T cells use for recognition of peptide-loaded MHC molecules is among the most sophisticated and versatile receptor systems in biology. It consists of specific and nonspecific transmembrane components which assemble to a functional signal transduction unit as the result of ligand binding. Correct assembly leads to activation and relocation of enzymes including membrane-associated, tyrosin-specific protein kinases and phosphatases. Transmembrane signaling in T cells depends on the correct assembly and cooperation among multiple molecular components. This may be related to a multitude of different cellular responses of T cells at different stages of differentiation, all elicited through the T cell receptor complex.     

Activation of JNK and p38 in rat hippocampus after kainic acid induced seizure

Kainic acid, an analogue of glutamate, causes limbic seizures and induces cell death in the rat brain. We examined the activation of MAPK family kinases; ERKs, JNKs and p38 kinase in rat hippocampus after KA treatment. Activation of all three kinases were observed at 30 min after the treatment, but, in contrary to ERK phosphorylation, which lasted up to 3 h, the phosphorylation of JNK and p38 returned to the basal level by 2 h. The phosphorylation of' upstream kinases for the MAPK family was distinct. The phosphorylation of MEK1 clearly increased at 30 min but diminished rapidly thereafter. The phosphorylation of MKK6 was also increased but reached peak at 2 h after KA treatment. However, the phosphorylation of other upstream kinases, SEK1 and MKK3, gradually decreased to 3 h after KA treatment. These results indicate that the KA activates all of the three MAPK family kinases with different time patterns and suggest the possibility that MKK3 and MKK6, and SEK1 may not be the upstream kinases for p38 and JNK in rat hippocampus.     

Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.