Protein Phosphorylation Mechanisms Research Articles

Prediction of Protein Kinase-Ligand Interactions through 2.5D Kinochemometrics.

So far, 518 protein kinases have been identified in the human genome. They share a common mechanism of protein phosphorylation and are involved in many critical biological processes of eukaryotic cells. Deregulation of the kinase phosphorylation function induces severe illnesses such as cancer, diabetes, or inflammatory diseases. Many actors in the pharmaceutical domain have made significant efforts to design potent and selective protein kinase inhibitors as new potential drugs. Because the ATP binding site is highly conserved in the protein kinase family, the design of selective inhibitors remains a challenge and has negatively impacted the progression of drug candidates to late-stage clinical development. The work presented here adopts a 2.5D kinochemometrics (KCM) approach, derived from proteochemometrics (PCM), in which protein kinases are depicted by a novel 3D descriptor and the ligands by 2D fingerprints. We demonstrate in two examples that the protein descriptor successfully classified protein kinases based on their group membership and their Asp-Phe-Gly (DFG) conformation. We also compared the performance of our models with those obtained from a full 2D KCM model and QSAR models. In both cases, the internal validation of the models demonstrated good capabilities to distinguish "active" from "inactive" protein kinase-ligand pairs. However, the external validation performed on two independent data sets showed that the two statistical models tended to overestimate the number of "inactive" pairs.

Phosphoproteome Analysis of Invasion and Metastasis-Related Factors in Pancreatic Cancer Cells.

Mechanisms of abnormal protein phosphorylation that regulate cell invasion and metastasis in pancreatic cancer remain obscure. In this study, we used high-throughput phosphorylation array to test two pancreatic cancer cell lines (PC-1 cells with a low, and PC-1.0 cells with a high potential for invasion and metastasis). We noted that a total of 57 proteins revealed a differential expression (fold change ≥ 2.0). Six candidate proteins were further validated by western blot with results found to be accordance with the array. Of 57 proteins, 32 up-regulated proteins (e.g. CaMK1-α and P90RSK) were mainly involved in ErbB and neurotrophin signaling pathways as determined using DAVID software, while 25 down-regulated proteins (e.g. BID and BRCA1) were closely involved in apoptosis and p53 signaling pathways. Moreover, four proteins (AKT1, Chk2, p53 and P70S6K) with different phosphorylation sites were found, not only among up-regulated, but also among down-regulated proteins. Importantly, specific phosphorylation sites can affect cell biological functions. CentiScaPe software calculated topological characteristics of each node in the protein-protein interaction (PPI) network: we found that AKT1 owns the maximum node degrees and betweenness in the up-regulation protein PPI network (26 nodes, average path length: 1.89, node degrees: 6.62±4.18, betweenness: 22.23±35.72), and p53 in the down-regulation protein PPI network (17 nodes, average path length: 2.04, node degrees: 3.65±2.47, betweenness: 16.59±29.58). In conclusion, the identification of abnormal protein phosphorylation related to invasion and metastasis may allow us to identify new biomarkers in an effort to develop novel therapeutic drug targets for pancreatic cancer treatment.

Effects of Phosphorylation of β Subunits of Phycocyanins on State Transition in the Model Cyanobacterium Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model cyanobacterium and has been used extensively for studies concerned with photosynthesis and environmental adaptation. Although dozens of protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted, only a few substrate proteins are known in Synechocystis. In this study, we report 194 in vivo phosphorylation sites from 149 proteins in Synechocystis, which were identified using a combination of peptide pre-fractionation, TiO(2) enrichment and liquid chromatograpy-tandem mass spectrometry (LC-MS/MS) analysis. These phosphorylated proteins are implicated in diverse biological processes, such as photosynthesis. Among all identified phosphoproteins involved in photosynthesis, the β subunits of phycocyanins (CpcBs) were found to be phosphorylated on Ser22, Ser49, Thr94 and Ser154. Four non-phosphorylated mutants were constructed by using site-directed mutagenesis. The in vivo characterization of the cpcB mutants showed a slower growth under high light irradiance and displayed fluorescence quenching to a lower level and less efficient energy transfer inside the phycobilisome (PBS). Notably, the non-phosphorylated mutants exhibited a slower state transition than the wild type. The current results demonstrated that the phosphorylation status of CpcBs affects the energy transfer and state transition of photosynthesis in Synechocystis. This study provides novel insights into the molecular mechanisms of protein phosphorylation in the regulation of photosynthesis in cyanobacteria and may facilitate the elucidation of the entire regulatory network by linking kinases to their physiological substrates.

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.

Computational identification of protein kinases and kinase-specific substrates in plants.

The protein phosphorylation catalyzed by protein kinases (PKs) plays an essential role in almost all biological progresses in plants. Thus, the identification of PKs and kinase-specific substrates is fundamental for understanding the regulatory mechanisms of protein phosphorylation especially in controlling plant growth and development. In this chapter, we describe the computational methods and protocols for the identification of PKs and kinase-specific substrates in plants, by using Vitis vinifera as an example. First, the proteome sequences and experimentally identified phosphorylation sites (p-sites) in Vitis vinifera were downloaded. The potential PKs were computationally identified based on preconstructed Hidden Markov Model (HMM) profiles and ortholog searches, whereas the kinase-specific p-sites, or site-specific kinase-substrate relations (ssKSRs) were initially predicted by the software package of Group-based Prediction System (GPS) and further processed by the iGPS algorithm (in vivo GPS) to filter potentially false positive hits. All primary data sets and prediction results of Vitis vinifera are available at: http://ekpd.biocuckoo.org/protocol.php.

Neuronal plasticity in hibernation and the proposed role of the microtubule-associated protein tau as a “master switch” regulating synaptic gain in neuronal networks

The present paper provides an overview of adaptive changes in brain structure and learning abilities during hibernation as a behavioral strategy used by several mammalian species to minimize energy expenditure under current or anticipated inhospitable environmental conditions. One cellular mechanism that contributes to the regulated suppression of metabolism and thermogenesis during hibernation is reversible phosphorylation of enzymes and proteins, which limits rates of flux through metabolic pathways. Reversible phosphorylation during hibernation also affects synaptic membrane proteins, a process known to be involved in synaptic plasticity. This mechanism of reversible protein phosphorylation also affects the microtubule-associated protein tau, thereby generating a condition that in the adult human brain is associated with aggregation of tau protein to paired helical filaments (PHFs), as observed in Alzheimer's disease. Here, we put forward the concept that phosphorylation of tau is a neuroprotective mechanism to escape NMDA-mediated hyperexcitability of neurons that would otherwise occur during slow gradual cooling of the brain. Phosphorylation of tau and its subsequent targeting to subsynaptic sites might, thus, work as a kind of "master switch," regulating NMDA receptor-mediated synaptic gain in a wide array of neuronal networks, thereby enabling entry into torpor. If this condition lasts too long, however, it may eventually turn into a pathological trigger, driving a cascade of events leading to neurodegeneration, as in Alzheimer's disease or other "tauopathies".

The Amplitude and Inactivation Properties of the Delayed Potassium Currents Are Regulated by Protein Kinase Activity in Hair Cells of the Frog Semicircular Canals

In hair cells dissected from the frog crista ampullaris, the combination of a calcium-dependent (IKCa) and a purely voltage-dependent component (IKV) gives rise to the delayed potassium current complex (IKD). These currents have been recently reported to display slow depolarization-induced inactivation and biphasic inactivation removal by hyperpolarization. The amplitude and inactivation kinetics of both IKCa and IKV are drastically modulated by a previously unrecognized mechanism of protein phosphorylation (sensitive to kinase inhibitors H89 and KT5823), which does not interfere with the transient potassium current (IA) or the calcium current (ICa). IKD amplitude was stable in cells patched with pipettes containing 8 mM ATP or under perforated-patch; under these conditions, a 10 min treatment with 10 µM H89 or 1–10 µM KT5823 reduced IKD amplitude by a mean of 67% at +40 mV. Similarly affected was the isolated IKV component (ICa blocked with Cd2+). Thus, a large potassium conductance can be activated by depolarization, but it is made available to the cell to a variable extent that depends on membrane potential and protein kinase activity. The total gKD ranged 4.6–44.0 nS in control cells, according to the level of steady-state inactivation, and was reduced to 1.4–2.7 nS after protein kinase inhibition. When sinusoidal membrane potential changes in the −70/−10 mV range were applied, to mimic receptor response to hair bundle deflection, IKD proved the main current dynamically activated and the only one regulated by PK: H89 decreased the total outward charge during each cycle by 60%. Phosphorylation appears to control both the amount of IKCa and IKV conductance activated by depolarization and the fraction thereof which can be rescued by removal of inactivation. The balance between the depolarizing transduction current and the repolarizing potassium current, and eventually the transmitter release at the cytoneural junction, are therefore modulated by a phosphorylation-mediated process.

Atomistic Insights Into the Regulatory Mechanisms Mediated by Post- Translational Modifications: Molecular Dynamics Investigations

Post-translational phosphorylation is a ubiquitous mechanism for cellular regulation, playing a crucial role in a diverse array of processes. At the molecular level, the phosphorylation seems to cause electrostatic perturbations which modulate the energy landscape governing protein folding, activity, protein-protein interactions and, conformational dynamics. However, details on structural effects induced by phosphorylation of proteins are still poorly understood. In this context, computational simulations recently provided a valuable alternative in the elucidation of principles and mechanisms of protein phosphorylation. In the present contribution, we review several recent studies devoted to molecular dynamics simulations of phospho-proteins with particular attention to proteins involved in the p53 pathway, ubiquitin pathway, protein kinases and intrinsically disordered proteins. Since application of biomolecular simulations to the investigation of mechanisms related to protein phospho-regulation is a newborn field for computational biology, our contribution could provide a suitable framework to plan future researches and to rationalize the available data in the field. Keywords: Molecular Dynamics Simulations, Phosphorylation, Conformational Change, Electrostatic Interaction, Kinase, p53, Ubiquitin, phosphorylation, loop-sheet-helix, Murine Double Minute clone 2

Atomistic Insights Into the Regulatory Mechanisms Mediated by Post- Translational Modifications: Molecular Dynamics Investigations

Post-translational phosphorylation is a ubiquitous mechanism for cellular regulation, playing a crucial role in a diverse array of processes. At the molecular level, the phosphorylation seems to cause electrostatic perturbations which modulate the energy landscape governing protein folding, activity, protein-protein interactions and, conformational dynamics. However, details on structural effects induced by phosphorylation of proteins are still poorly understood. In this context, computational simulations recently provided a valuable alternative in the elucidation of principles and mechanisms of protein phosphorylation. In the present contribution, we review several recent studies devoted to molecular dynamics simulations of phospho-proteins with particular attention to proteins involved in the p53 pathway, ubiquitin pathway, protein kinases and intrinsically disordered proteins. Since application of biomolecular simulations to the investigation of mechanisms related to protein phospho-regulation is a newborn field for computational biology, our contribution could provide a suitable framework to plan future researches and to rationalize the available data in the field.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Using multitask classification methods to investigate the kinase-specific phosphorylation sites

BackgroundIdentification of phosphorylation sites by computational methods is becoming increasingly important because it reduces labor-intensive and costly experiments and can improve our understanding of the common properties and underlying mechanisms of protein phosphorylation.MethodsA multitask learning framework for learning four kinase families simultaneously, instead of studying each kinase family of phosphorylation sites separately, is presented in the study. The framework includes two multitask classification methods: the Multi-Task Least Squares Support Vector Machines (MTLS-SVMs) and the Multi-Task Feature Selection (MT-Feat3).ResultsUsing the multitask learning framework, we successfully identify 18 common features shared by four kinase families of phosphorylation sites. The reliability of selected features is demonstrated by the consistent performance in two multi-task learning methods.ConclusionsThe selected features can be used to build efficient multitask classifiers with good performance, suggesting they are important to protein phosphorylation across 4 kinase families.

Crystallization and preliminary X-ray analysis of the stress-response PPM phosphatase RsbX from Bacillus subtilis.

RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase sigma factor SigB. In order to elucidate the structural-functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 angstrom resolution with an R(merge) of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 angstrom , alpha = 98.8, beta = 90.0, gamma = 108.4 degrees.

PL-02-02: Amyloid precursor sorting and processing: Regulation by transmitters, hormones, and protein phosphorylation mechanisms

PL-02-02: Amyloid precursor sorting and processing: Regulation by transmitters, hormones, and protein phosphorylation mechanisms

A Novel Rice MAPK Gene,OsBIMK2,is Involved in Disease‐Resistance Responses

The mitogen-activated protein kinase (MAPK) cascades play important roles in transmission of extracellular signals to the downstream effector proteins through a mechanism of protein phosphorylation. In this study, we isolated and identified a novel rice MAPK gene, OSBIMK2 ( ORYZAE SATIVA L. BTH-Induced MAP Kinase 2). The OSBIMK2 encodes a 506 amino acid protein with molecular weight of 63 kD. The recombinant OSBIMK2 expressed in ESCHERICHIA COLI showed an autophosphorylation activity IN VITRO. OSBIMK2 is a single-copy gene in the rice genome. Expression of OSBIMK2 was activated upon treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice. Expression of OsBIMK2 was also up-regulated during early stage after inoculation with MAGNAPORTHE GRISEA in BTH-treated rice seedlings and during an incompatible interaction between M. GRISEA and a blast-resistant rice genotype. Over-expression of the rice OSBIMK2 gene in transgenic tobacco resulted in an enhanced disease resistance against tomato mosaic virus and a fungal pathogen, ALTERNARIA ALTERNATA. These results suggest that OSBIMK2 plays a role in disease resistance responses.

Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).

Characterizing the Microenvironment Surrounding Phosphorylated Protein Sites

Protein phosphorylation plays an important role in various cellular processes. Due to its high complexity, the mechanism needs to be further studied. In the last few years, many methods have been contributed to this field, but almost all of them investigated the mechanism based on protein sequences around protein sites. In this study, we implement an exploration by characterizing the microenvironment surrounding phosphorylated protein sites with a modified shell model, and obtain some significant properties by the rank-sum test, such as the lack of some classes of residues, atoms, and secondary structures. Furthermore, we find that the depletion of some properties affects protein phosphorylation remarkably. Our results suggest that it is a meaningful direction to explore the mechanism of protein phosphorylation from microenvironment and we expect further findings along with the increasing size of phosphorylation and protein structure data.

Phosphorylation of AMPA Receptors: Mechanisms and Synaptic Plasticity

The ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.

The role of Krs1 in cell cycle arrest.

The Krs1 (kinase responsive to stress 1) kinase has been proposed to mediate signals initiated by various forms of cellular or environmental stress for the process of growth arrest and apoptosis. The functional role of Krs1 in cell growth arrest may involve a constant, stress-independent proteolytic modification of the full-length kinase p63(Krs1), via caspase-like activity, to produce kinase-active kinase fragment, p33(Krs1). Induction of the kinase activity of p33(Krs1) is closely correlated with cell growth arrest in the G(0)/G(1) phase due to serum starvation, contact inhibition or growth-arresting agents. Deactivation of p33(Krs1) is closely associated with cell entry into the cell cycle. In response to stress shock the conversion of p63(Krs1) to p33(Krs1) is enhanced, and the kinase activity of p33(Krs1) is additionally increased in quiescent cells that undergo apoptosis. Both mechanisms of proteolytic modification and protein phosphorylation are involved in a complex control of the kinase activity and function of p33(Krs1) in response to environmental changes leading to cell growth arrest in quiescence or quiescence-related apoptosis. Numerous studies have shown that signaling pathways in malignantly transformed cells are regulated differently from their counterpart pathways in normal counterpart cells in response to stress shock or anticancer agents. It is desirable to develop one type of anticancer agent to selectively growth-arrest normal counterpart cells in G(0)/G(1) phase of the cell cycle through the control of Krs1-involved signaling pathways. The success of arresting normal cells in a quiescent state will provide access for other types of anticancer treatments to induce cell death of cancerous cells, which are resistant to growth arrest in other phases of the cell cycle.

Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes

It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures. This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S- rolB and 35S- rolC genes in comparison with a non-transformed callus culture. The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells. Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R. cordifolia calluses, whereas ethephon did not. A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only. We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.

Protein Kinase C-Stimulated Formation of Ethanolamine from Phosphatidylethanolamine Involves a Protein Phosphorylation Mechanism: Negative Regulation by p21 Ras Protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-α (PKC-α)-activated, presently unknown, mechanism. Now we report that addition of a PKC-α-enriched purified PKC preparation or recombinant PKC-α to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly (∼6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-α, ATP, and PMA on [14C]PtdEtn hydrolysis were inhibited by GF 109203X (10 μM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller (∼3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-α is inhibited by p21 Ras.