MAP kinases: universal multi-purpose signaling tools

MAP (mitogen-activated protein) kinases are serine/threonine protein kinases and mediate intracellular phosphorylation events linking various extracellular signals to different cellular targets. MAP kinase, MAP kinase kinase and MAP kinase kinase kinase are functional protein kinase units that are conserved in several signal transduction pathways in animals and yeasts. Isolation of all three components was also shown in plants and suggests conservation of a protein kinase module in all eukaryotic cells. In plants, MAP kinase modules appear to be involved in ethylene signaling and auxin-induced cell proliferation. Therefore, coupling of different extracellular signals to different physiological responses is mediated by MAP kinase cascades and appears to have evolved from a single prototypical protein kinase module which has been adapted to the specific requirements of different organisms.     

疱疹病毒蛋白激酶功能的研究进展

丝氨酸／苏氨酸激酶存在于所有已知的疱疹病毒中,它们具有多种功能,参与病毒感染的整个过程,尤其是病毒与机体的相互作用。主要阐述了两类保守的疱疹病毒丝氨酸／苏氨酸激酶（单纯疱疹病毒HSV的ULl3激酶和US3激酶）在病毒感染过程的重要作用。两者都参与调控细胞和病毒基因的表达,介导病毒衣壳出核以及免疫逃避。虽然这些激酶对病毒在体外培养细胞中复制的影响各不相同,但是对于病毒的毒力非常重要,因此,可用作抗病毒药物设计的靶位。     

Approach to systematic analysis of serine/threonine phosphoproteome using Beta elimination and subsequent side effects: intramolecular linkage and/or racemisation

Complete analysis of the phosphorylation of serine and threonine residues directly from biological extracts is still at an early stage and will remain a challenging goal for many years. Analysis of phosphorylated proteins and identification of the phosphorylated sites in a crude biological extract is a major topic in proteomics, since phosphorylation plays a dominant role in post-translational protein modification. Beta elimination of the serine/threonine-bound phosphate by alkali action generates (methyl)dehydroalanine. The reactivity of this group susceptible of nucleophilic attacks might be used as a tool for phosphoproteome analysis. Most of the known serine/threonine kinases recognize motifs in protein targets that are rich in lysine(s) and/or arginine(s). The (methyl)dehydroalanine resulting from beta elimination of the serine/threonine-bound phosphate by alkali action is likely to react with the amino groups of these neighboring amino acids. Furthermore, the addition reaction of dehydroalanine-peptides with a nucleophilic group more likely generates diastereoisomers derivatives. The internal cyclic bonds and/or the stereoisomer peptide derivatives thus generated confer resistance to trypsin cleavage and/or constitute stop signals for exopeptidases such as carboxypeptidase. This might form the basis of a method to facilitate the systematic identification of phosphorylated peptides.     

Structure–sequence features based prediction of phosphosites of serine/threonine protein kinases of Mycobacterium tuberculosis

Elucidation of signaling events in a pathogen is potentially important to tackle the infection caused by it. Such events mediated by protein phosphorylation play important roles in infection, and therefore, to predict the phosphosites and substrates of the serine/threonine protein kinases, we have developed a Machine learning-based approach for Mycobacterium tuberculosis serine/threonine protein kinases using kinase-peptide structure–sequence data. This approach utilizes features derived from kinase three-dimensional-structure environment and known phosphosite sequences to generate support vector machine (SVM)-based kinase-specific predictions of phosphosites of serine/threonine protein kinases (STPKs) with no or scarce data of their substrates. SVM outperformed the four machine learning algorithms we tried (random forest, logistic regression, SVM, and k-nearest neighbors) with an area under the curve receiver-operating characteristic value of 0.88 on the independent testing dataset and a 10-fold cross-validation accuracy of ~81.6% for the final model. Our predicted phosphosites of M. tuberculosis STPKs form a useful resource for experimental biologists enabling elucidation of STPK mediated posttranslational regulation of important cellular processes.     

Classification of common functional loops of kinase super-families

A structural classification of loops has been obtained from a set of 141 protein structures classified as kinases. A total of 1813 loops was classified into 133 subclasses (9 betabeta(links), 15 betabeta(hairpins), 31 alpha-alpha, 46 alpha-beta and 32 beta-alpha). Functional information and specific features relating subclasses and function were included in the classification. Functional loops such as the P-loop (shared by different folds) or the Gly-rich-loop, among others, were classified into structural motifs. As a result, a common mechanism of catalysis and substrate binding was proved for most kinases. Additionally, the multiple-alignment of loop sequences made within each subclass was shown to be useful for comparative modeling of kinase loops. The classification is summarized in a kinase loop database located at http://sbi.imim.es/archki.     

Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites

Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within‐Subfamily, (iii) Within‐Family, (iv) Within‐Group, and (v) Across‐Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low‐magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within‐Subfamily and Within‐family) than in distant ones (Within‐Group and Across‐Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue‐wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin‐dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein‐protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957–978. © 2016 Wiley Periodicals, Inc.     

Cold acclimation of SnRK2.2 kinases mutant Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, plant‐specific serine/threonine kinases (SnRK2 kinases) play a central role in sulfur metabolism. However, their role in environmental stress has not been clearly understood. Cold stress is one of the most important factors that limit the growth, productivity, and development of photosynthetic organisms. In this report, the effect of cold stress on some physiological parameters were investigated in SnRK2.2 mutant and wild type C. reinhardtii culture. Our results showed that cold stress had significantly enhanced lipid peroxidation rate in wild type, while no significant change was observed in SnRK2.2 mutant culture. Our data also indicated a decline in Rubisco protein amount of wild type culture under low temperature exposure. Low temperature reduced glutathione reductase (GR) activity in the wild type, while GR activity was enhanced in SnRK2.2 mutant. This result indicated the potential of SnRK2 kinase in cold acclimation process.     

Computational proteomics of biomolecular interactions in the sequence and structure space of the tyrosine kinome: deciphering the molecular basis of the kinase inhibitors selectivity

Understanding and predicting the molecular basis of protein kinases specificity against existing therapeutic agents remains highly challenging and deciphering this complexity presents an important problem in discovery and development of effective cancer drugs. We explore a recently introduced computational approach for in silico profiling of the tyrosine kinases binding specificity with a class of the pyrido-[2,3-d]pyrimidine kinase inhibitors. Computational proteomics analysis of the ligand-protein interactions using parallel simulated tempering with an ensemble of the tyrosine kinases crystal structures reveals an important molecular determinant of the kinase specificity. The pyrido-[2,3-d]pyrimidine inhibitors are capable of dynamically interacting with both active and inactive forms of the tyrosine kinases, accommodating structurally different kinase conformations with a similar binding affinity. Conformational tolerance of the protein tyrosine kinases binding with the pyrido[2,3-d]pyrimidine inhibitors provides the molecular basis for the broad spectrum of potent activities and agrees with the experimental inhibition profiles. The analysis of the pyrido[2,3-d]pyrimidine sensitivities against a number of clinically relevant ABL kinase mutants suggests an important role of conformational adaptability of multitargeted kinase inhibitors in developing drug resistance mechanisms. The presented computational approach may be useful in complementing proteomics technologies to characterize activity signatures of small molecules against a large number of potential kinase targets.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.     

High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

Fast and automated functional classification with MED‐SuMo: An application on purine‐binding proteins

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED‐SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED‐SMA is an automated and fast method to classify binding sites. It is based on MED‐SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora‐A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED‐SMA approach is demonstrated as it gathers binding sites of proteins with similar structure‐activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target‐based drug design and prediction of cross‐reactivity and therefore potential toxic side effects.     

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X‐ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate‐binding site. ROP kinases contain a typical bilobate kinase fold and a novel N‐terminal extension that both stabilizes the N‐lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N‐terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N‐terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP‐binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.     

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASK), ASKetha (ASK) and ASKiota (ASK). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASK is expressed in the whole embryo during its development, ASK expression is limited to the suspensor cells. No signal was detected for ASK, ASK and ASK in developing embryos.     

粗糙脉孢菌丝氨酸/苏氨酸激酶家族基因敲除库纤维素酶表达分泌研究

【目的】蛋白磷酸化在丝状真菌细胞对外界纤维素酶诱导信号感应以及信号胞内的传导过程中有着重要的作用,而蛋白磷酸化是由蛋白激酶来完成的。为了挖掘在丝状真菌纤维素酶表达过程中发挥重要作用的激酶基因,对粗糙脉孢菌丝氨酸/苏氨酸家族的61株蛋白激酶单基因突变体的纤维素酶表达分泌情况进行了分析测定。【方法】在以微晶纤维素为唯一碳源的条件下,7株单基因突变体胞外分泌蛋白产量有显著变化,随后,对这7株突变体胞外蛋白进行了详细的SDS-PAGE分析和内切-β-1,4-葡聚糖酶酶活、β-葡萄糖苷酶酶活、外切纤维素酶酶活以及木聚糖酶酶活的测定。【结果】突变株W14、W38、W87和W40胞外分泌蛋白含量提高了30%以上,除了突变株W14外,其它突变体的内切-β-1,4-葡聚糖酶酶活分别显著提高了62%、42%和42%。而突变株W85、W26和W46胞外分泌蛋白含量降低了50%以上,相对应的内切-β-1,4-葡聚糖酶酶活也分别下降了86%、75%和84%。【结论】这些关于粗糙脉孢菌丝氨酸/苏氨酸家族蛋白激酶基因的挖掘,为进一步深入研究蛋白激酶在纤维素酶诱导表达调控中的分子机理奠定了基础。     

Haspin-like proteins: a new family of evolutionarily conserved putative eukaryotic protein kinases

Haspin (haploid germ cell-specific nuclear protein kinase) is reported to be a serine/threonine kinase that may play a role in cell-cycle cessation and differentiation of haploid germ cells. In addition, Haspin mRNA can be detected in diploid cell lines and tissues. Here, Haspin-like proteins are identified in several major eukaryotic phyla-including yeasts, plants, flies, fish, and mammals-and an extended group in Caenorhabditis elegans. The Haspin-like proteins have a complete but divergent eukaryotic protein kinase domain sequence. Although clearly related to one another and to other eukaryotic protein kinases, the Haspin-related proteins lack conservation of a subset of residues that are almost invariant in known kinases and possess distinctive inserted regions. In fact, phylogenetic analysis indicates that the Haspin-like proteins form a novel eukaryotic protein kinase family distinct from those previously defined. The identification of related proteins in model organisms provides some initial insight into their functional properties and will provide new experimental avenues by which to determine the function of the Haspin proteins in mammalian cells.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.