Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling

Some apicomplexan parasites have evolved distinct protein kinase families to modulate host cell structure and function. Toxoplasma gondii rhoptry protein kinases and pseudokinases are involved in virulence and modulation of host cell signalling. The proteome of Plasmodium falciparum contains a family of putative kinases called FIKKs, some of which are exported to the host red blood cell and might play a role in erythrocyte remodelling. In this review we will discuss kinases known to be critical for host cell invasion, intracellular growth and egress, focusing on (i) calcium-dependent protein kinases and (ii) the secreted kinases that are unique to Toxoplasma (rhoptry protein kinases and pseudokinases) and Plasmodium (FIKKs)

Tv-RIO1 – an atypical protein kinase from the parasitic nematode Trichostrongylus vitrinus

Background: Protein kinases are key enzymes that regulate a wide range of cellular processes, including cell-cycle progression, transcription, DNA replication and metabolic functions. These enzymes catalyse the transfer of phosphates to serine, threonine and tyrosine residues, thus playing functional roles in reversible protein phosphorylation. There are two main groups, namely eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs); RIO kinases belong to the latter group. While there is some information about RIO kinases and their roles in animals, nothing is known about them in parasites. This is the first study to characterise a RIO1 kinase from any parasite. Results: A full-length cDNA (Tv-rio-1) encoding a RIO1 protein kinase (Tv-RIO1) was isolated from the economically important parasitic nematode Trichostrongylus vitrinus (Order Strongylida). The uninterrupted open reading frame (ORF) of 1476 nucleotides encoded a protein of 491 amino acids, containing the characteristic RIO1 motif LVHADLSEYNTL. Tv-rio-1 was transcribed at the highest level in the third-stage larva (L3), and a higher level in adult females than in males. Comparison with homologues from other organisms showed that protein Tv-RIO1 had significant homology to related proteins from a range of metazoans and plants. Amino acid sequence identity was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic analyses of selected amino acid sequence data revealed Tv-RIO1 to be most closely related to the proteins in the species of Caenorhabditis. A structural model of Tv-RIO1 was constructed and compared with the published crystal structure of RIO1 of Archaeoglobus fulgidus (Af-Rio1). Conclusion: This study provides the first insights into the RIO1 protein kinases of nematodes, and a foundation for further investigations into the biochemical and functional roles of this molecule in biological processes in parasitic nematodes

Positional information readout in Ca2+Ca^{2+} signaling

Living cells respond to spatial signals. Signal transmission to the cell interior often involves the release of second messengers like $Ca^{2+}$ . They will eventually trigger a physiological response by activating kinases that in turn activate target proteins through phosphorylation. Here, we investigate theoretically how positional information can be accurately read out by protein phosphorylation in spite of rapid second messenger diffusion. We find that accuracy is increased by binding of the kinases to the cell membrane prior to phosphorylation and by increasing the rate of $Ca^{2+}$ loss from the cell interior. These findings could explain some salient features of conventional protein kinases C

MAP3Kinase-dependent SnRK2-kinase activation is required for abscisic acid signal transduction and rapid osmotic stress response.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction

Extracellular signal-regulated kinases mediate the enhancing effects of inflammatory mediators on resurgent currents in dorsal root ganglion neurons

Previously we reported that a group of inflammatory mediators significantly enhanced resurgent currents in dorsal root ganglion neurons. To understand the underlying intracellular signaling mechanism, we investigated the effects of inhibition of extracellular signal-regulated kinases and protein kinase C on the enhancing effects of inflammatory mediators on resurgent currents in rat dorsal root ganglion neurons. We found that the extracellular signal-regulated kinases inhibitor U0126 completely prevented the enhancing effects of the inflammatory mediators on both Tetrodotoxin-sensitive and Tetrodotoxin-resistant resurgent currents in both small and medium dorsal root ganglion neurons. U0126 substantially reduced repetitive firing in small dorsal root ganglion neurons exposed to inflammatory mediators, consistent with prevention of resurgent current amplitude increases. The protein kinase C inhibitor Bisindolylmaleimide I also showed attenuating effects on resurgent currents, although to a lesser extent compared to extracellular signal-regulated kinases inhibition. These results indicate a critical role of extracellular signal-regulated kinases signaling in modulating resurgent currents and membrane excitability in dorsal root ganglion neurons treated with inflammatory mediators. It is also suggested that targeting extracellular signal-regulated kinases-resurgent currents might be a useful strategy to reduce inflammatory pain

Protein Kinases

Proteins are the work horses of the cell. As regulators of protein function, protein kinases are involved in the control of cellular functions via intricate signalling pathways, allowing for fine tuning of physiological functions. This book is a collaborative effort, with contribution from experts in their respective fields, reflecting the spirit of collaboration - across disciplines and borders - that exists in modern science. Here, we review the existing literature and, on occasions, provide novel data on the function of protein kinases in various systems. We also discuss the implications of these findings in the context of disease, treatment, and drug development

Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A)

Plants have to respond and adapt to a variety of continuously changing environmental factors in order to establish an appropriate developmental strategy to ensure survival. There are ample data showing that protein phosphorylation/dephosphorylation plays a central role in cellular signal transduction in all organisms (Herrmann et al. 2006; Stone and Walker 1995). Interestingly, plants have a similar system as mammals to detect and transfer signals across the cell membrane into the nucleus where adaptations could be initiated. For the detection and transfer of an external signal, mammalian systems have receptor protein kinases. The proteins contain a single hydrophobic transmembrane domain, an extracellular domain and protein kinase domain. The majority of animal receptor kinases are phosphorylated on tyrosine residues within the kinase domain (receptor tyrosin kinases or RTKs; Ullrich and Schlessinger 1990), but a few were discovered which are phosphorylated on serine and threonine residues (Lin et al. 1992). In plants, two different types of transmembrane receptor kinases are known, including receptor-like serine/threonine (Ser/Thr) kinases (receptor-like kinases RLKs; Shiu and Bleecker 2001, 2003; Shiu et al. 2004; Walker 1994), structurally similar to mammalian RTKs, and receptor histidine (His) kinases (Grefen and Harter 2004; Mizuno 2005; Urao et al. 2000). Since the first RLK-encoding gene family was found in Zea mays (Walker and Zhang 1990), thousands of RLK genes have been identified from many different plant species. The Arabidopsis genome contains more than 600 members, representing nearly 2.5% of the annotated protein-coding genes; and more than 1000 members were annotated in the rice genome (Shiu et al. 2004). Approximately 25% of the Arabidopsis RLKs contain only a kinase domain with no apparent signal sequence or transmembrane region and thus were collectively named as receptor like cytoplasmic kinases (RLCKs). Arabidopsis RLCKs can be subdivided into 12 classes with 193 protein coding genes all together. Concerning the function of plant RLCKs, at the present only few members have been characterized and it is very likely that they play major role in the perception and 93 transmission of external signals perceived by RLKs (Zhou et al. 1995; Murase et al. 2004). Recently, our group as well as a group in Germany reported a direct interaction of plant ROP GTPases with receptor-like cytoplasmic kinases (RLCK class VI) from Arabidopsis (Molendijk et al. 2008) and alfalfa (Dorjgotov et al. 2009). Moreover, we provide evidences that kinases belonging to the RLCK Class VI family of Medicago truncatula and Arabidopsis thaliana can be specifically activated by GTP-bound ROP GTPases in vitro further supporting the view that plant Rho (ROP) G-proteins may directly regulate downstream kinase signaling. A further kinase designated as cysteine-rich receptor kinase (NCRK) belonging to a distinct kinase family has also been shown to interact with ROPs (Molendijk et al. 2008). None of these plant specific ROP-interacting kinases has any characteristic domain or motif that could be correlated with their ability to bind ROP GTPases. Plant specific ROP GTPases are versatile molecular switches in many processes during plant growth, development and responses to the environment and thus a possible implication of RLCKs in these ROP-dependent signal transduction pathways is in discussion. Our general aim was to characterize the members of the Arabidopsis thaliana RLCK Class VI family of protein kinases. Despite of their potential significance in ROP GTPase mediated signaling, hardly any functional information was available until now about the fourteen Arabidopsis RLCK Class VI members. Sequence comparison and phylogenetic analysis revealed that gene duplication played a significant role in the formation of this kinase family and allowed the separation of the 14 RCLK VI kinases into two groups with seven members each (A1 to A7 and B1 to B7). The proteins are highly homologous to each other, especially in the kinase domain, but are divergent from the related kinase families. It was established that, several members have an N-terminal UspA (“universal stress protein”) domain (group B members) or an N-terminal serine-rich region (group A members). In order to formulate a possible biological role of AtRLCK_VI kinases, real-time quantitative reverse transcription-polymerase reaction (qRT-PCR) was used to determine relative transcript levels in the various organs (root, rosette leaves, cauline leaves, 94 inflorescence stem, flower buds, open flowers, siliques. exponentially dividing cultured cells) of the Arabidopsis plant as well as under a series of abiotic stress/hormone (osmotic, sugar, salt stress, oxidative stress, cold and hormone treatment) treatments in seedlings. AtRLCK VI genes exhibited diverse expression patterns in the various plant organs as well as in response to stress/hormone treatments..

c-Jun N-Terminal Kinase in Inflammation and Rheumatic Diseases.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are activated by environmental stress. JNK is also activated by proinflammatory cytokines, such as TNF and IL-1, and Toll-like receptor ligands. This pathway, therefore, can act as a critical convergence point in immune system signaling for both adaptive and innate responses. Like other MAPKs, the JNKs are activated via the sequential activation of protein kinases that includes two dual-specificity MAP kinase kinases (MKK4 and MKK7) and multiple MAP kinase kinase kinases. MAPKs, including JNKs, can be deactivated by a specialized group of phosphatases, called MAP kinase phosphatases. JNK phosphorylates and regulates the activity of transcription factors other than c-Jun, including ATF2, Elk-1, p53 and c-Myc and non-transcription factors, such as members of the Bcl-2 family. The pathway plays a critical role in cell proliferation, apoptosis, angiogenesis and migration. In this review, an overview of the functions that are related to rheumatic diseases is presented. In addition, some diseases in which JNK participates will be highlighted

Protein phosphorylation in yeast mitochondria

We describe the identification and submitochondrial localization of four protein kinases and of their target proteins in derepressed yeast mitochondria. The activity of one of the kinases depends on the presence of cyclic AMP (cAMP). It is soluble and localized in the mitochondrial intermembrane space. Its natural target is a polypeptide of 40 kDa molecular mass, which is bound to the inner membrane. Besides this natural target this kinase phosphorylates acidic heterologous proteins, like casein, with high efficiency. The other protein kinases identified so far are cAMP-independent. At least one is localized in the matrix having its natural substrates (49 and 24 kDa) in the same compartment. Two others are firmly bound to the inner membrane phosphorylating target proteins in the inner membrane (52·5 kDa) and in the intermembrane space (17·5 kDa), respectively

MoKCa database - mutations of kinases in cancer

Members of the protein kinase family are amongst the most commonly mutated genes in human cancer, and both mutated and activated protein kinases have proved to be tractable targets for the development of new anticancer therapies The MoKCa database (Mutations of Kinases in Cancer, http://strubiol.icr.ac.uk/extra/mokca) has been developed to structurally and functionally annotate, and where possible predict, the phenotypic consequences of mutations in protein kinases implicated in cancer. Somatic mutation data from tumours and tumour cell lines have been mapped onto the crystal structures of the affected protein domains. Positions of the mutated amino-acids are highlighted on a sequence-based domain pictogram, as well as a 3D-image of the protein structure, and in a molecular graphics package, integrated for interactive viewing. The data associated with each mutation is presented in the Web interface, along with expert annotation of the detailed molecular functional implications of the mutation. Proteins are linked to functional annotation resources and are annotated with structural and functional features such as domains and phosphorylation sites. MoKCa aims to provide assessments available from multiple sources and algorithms for each potential cancer-associated mutation, and present these together in a consistent and coherent fashion to facilitate authoritative annotation by cancer biologists and structural biologists, directly involved in the generation and analysis of new mutational data