Human members of the eukaryotic protein kinase family.
ABSTRACT: BACKGROUND:Eukaryotic protein kinases (EPKs) constitute one of the largest recognized protein families represented in the human genome. EPKs, which are similar to each other in sequence, structure and biochemical properties, are important players in virtually every signaling pathway involved in normal development and disease. Near completion of projects to sequence the human genome and transcriptome provide an opportunity to identify and perform sequence analysis on a nearly complete set of human EPKs. RESULTS:Publicly available genetic sequence data were searched for human sequences that potentially represent EPK family members. After removal of duplicates, splice variants and pseudogenes, this search yielded 510 sequences with recognizable similarity to the EPK family. Protein sequences of putative EPK catalytic domains identified in the search were aligned, and a phonogram was constructed based on the alignment. Representative sequence records in GenBank were identified, and derived information about gene mapping and nomenclature was summarized. CONCLUSIONS:This work represents a nearly comprehensive census and early bioinformatics overview of the EPKs encoded in the human genome. Evaluation of the sequence relationships between these proteins contributes contextual information that enhances understanding of individual family members. This curation of human EPK sequences provides tools and a framework for the further characterization of this important class of enzymes.
Project description:BACKGROUND: Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism. RESULTS: Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in P. falciparum and just one member in some other Apicomplexan species. CONCLUSION: The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells.
Project description:Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The Schistosoma mansoni genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the S. mansoni predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets.We have identified 252 ePKs, which corresponds to 1.9% of the S. mansoni predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that S. mansoni has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in S. mansoni or belong to an expanded family in this parasite. Only 16 S. mansoni ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite.Our approach has improved the functional annotation of 40% of S. mansoni ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of S. mansoni in response to diverse environments during the parasite development, vector interaction, and host infection.
Project description:Protein kinases have been implicated in the regulation of many processes that guide pathogen development throughout the course of infection. A survey of the Sclerotinia sclerotiorum genome for genes encoding proteins containing the highly conserved eukaryotic protein kinase (ePK) domain, the largest protein kinase superfamily, revealed 92 S.?sclerotiorum ePKs. This review examines the composition of the S.?sclerotiorum ePKs based on conserved motifs within the ePK domain family, and relates this to orthologues found in other filamentous fungi and yeasts. The ePKs are also discussed in terms of their proposed role(s) in aspects of host pathogenesis, including the coordination of mycelial growth/development and deployment of pathogenicity determinants in response to environmental stimuli, nutrients and stress.
Project description:The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.
Project description:The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases, though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one subset of a diverse superfamily of enzymes built on a common protein kinase-like (PKL) fold. We explored this huge phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20 highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison between and within families revealed ten key residues that are highly conserved across families. However, all but one of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution.
Project description:Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified "Shell" that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.
Project description:Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate a myriad of signaling events. It has been postulated that EPKs' active and inactive states depend on the structural architecture of their hydrophobic cores, organized around two highly conserved spines: C-spine and R-spine. How the spines orchestrate the transition of the enzyme between catalytically uncommitted and committed states remains elusive. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we found that the hydrophobic core of the catalytic subunit of protein kinase A, a prototypical and ubiquitous EPK, moves synchronously to poise the C subunit for catalysis in response to binding adenosine 5'-triphosphate. In addition to completing the C-spine, the adenine ring fuses the ? structures of the N-lobe and the C-lobe. Additional residues that bridge the two spines (I150 and V104) are revealed as part of the correlated hydrophobic network; their importance was validated by mutagenesis, which led to inactivation. Because the hydrophobic architecture of the catalytic core is conserved throughout the EPK superfamily, the present study suggests a universal mechanism for dynamically driven allosteric activation of kinases mediated by coordinated signal transmission through ordered motifs in their hydrophobic cores.
Project description:Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.
Project description:The BLAST-Like Alignment Tool (BLAT) is used to find genomic sequences that match a protein or DNA sequence submitted by the user. BLAT is typically used for searching similar sequences within the same or closely related species. It was developed to align millions of expressed sequence tags and mouse whole-genome random reads to the human genome at a higher speed. It is freely available either on the Web or as a downloadable stand-alone program. BLAT search results provide a link for visualization in the University of California, Santa Cruz (UCSC) Genome Browser, where associated biological information may be obtained. Three example protocols are given: using an mRNA sequence to identify the exon-intron locations and associated gene in the genomic sequence of the same species, using a protein sequence to identify the coding regions in a genomic sequence and to search for gene family members in the same species, and using a protein sequence to find homologs in another species.
Project description:Although conserved noncoding elements (CNEs) constitute the majority of sequences under purifying selection in the human genome, they remain poorly understood. CNEs seem to be largely unique, with no large families of similar elements reported to date. Here, we search for CNEs among the ancestral repeat classes in the human genome and report the discovery of a large CNE family containing >900 members. This family belongs to the MER121 class of repeats. Although the MER121 family members show considerable sequence variation among one another, the individual copies show striking conservation in orthologous locations across the human, dog, mouse, and rat genomes. The element is also present and conserved in orthologous locations in the marsupial, but its genome-wide dispersal postdates the divergence from birds. The comparative genomic data indicate that MER121 does not encode a family of either protein-coding or RNA genes. Although the precise function of these elements remains unknown, the evidence suggests that this unusual family may play a cis-regulatory or structural role in mammalian genomes.