Large and diverse numbers of human diseases with HIKE mutations

HIKE is a highly conserved sequence motif identified as a candidate pleckstrin-homology (PH) domain binding site in Gbeta proteins, protein kinases, ankyrin and kinesin. HIKE motifs occur also in gelsolin, neurogranin, neuromodulin and in the PH domain of Bruton tyrosin kinase (BTK). Phosphatidylinositol-binding sequences more distantly related to HIKE are present in gelsolin, in the G protein-coupled receptor kinase 4 and in Trop-2. HIKE regions have been demonstrated to bind both proteins and lipids, and to regulate the interaction of Gbeta, neuromodulin and the BTK PH domain with downstream effectors and the cell membrane. Remarkably, mutations of the HIKE regions are common in diverse human genetic diseases. Several HIKE mutations in protein kinases lead to constitutive activation and cellular transformation, e.g. in MEN-2B, acute myeloid and mast cell leukemias, hereditary papillary renal carcinomas and multiple myeloma. Kinase-inactivating HIKE mutations cause Hirschsprung's disease, piebaldism, insulin resistance and developmental dysplasias. HIKE mutations in the PH domain of BTK lead to X-linked agammaglobulinemia, and different forms of amyloidosis are caused by mutations of HIKE-bearing molecules, for example gelsolin, Ret and Trop-2. Thus, quite diverse genetic diseases might share common molecular mechanisms. These include altered interactions of the mutated molecules with downstream effectors or the cell membrane, and defects in intracellular transport.     

Prediction of the Repeat Domain Structures and Impact of Parkinsonism‐Associated Variations on Structure and Function of all Functional Domains of Leucine‐Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine‐rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras‐like GTPase (ROC) domain and C‐terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD‐causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three‐dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three‐dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three‐dimensional structures of repeat domains of other proteins, the tandem Roc–COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein–protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc–COR and kinase domains, and (3) the effects of some PD‐associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.     

The SH3 Domain of Bruton's Tyrosine Kinase Interacts with Vav, Sam68 and EWS

Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro → Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.     

Multiple Functional Effects of RET Kinase Domain Sequence Variants in Hirschsprung Disease

The REarranged during Transfection (RET) gene encodes a receptor tyrosine kinase required for maturation of the enteric nervous system. RET sequence variants occur in the congenital abnormality Hirschsprung disease (HSCR), characterized by absence of ganglia in the intestinal tract. Although HSCR‐RET variants are predicted to inactivate RET, the molecular mechanisms of these events are not well characterized. Using structure‐based models of RET, we predicted the molecular consequences of 23 HSCR‐associated missense variants and how they lead to receptor dysfunction. We validated our predictions in biochemical and cell‐based assays to explore mutational effects on RET protein functions. We found a minority of HSCR‐RET variants abrogated RET kinase function, while the remaining mutants were phosphorylated and transduced intracellular signals. HSCR‐RET sequence variants also impacted on maturation, stability, and degradation of RET proteins. We showed that each variant conferred a unique combination of effects that together impaired RET protein activity. However, all tested variants impaired RET‐mediated cellular functions, including cell transformation and migration. Our data indicate that the molecular mechanisms of impaired RET function in HSCR are highly variable. Although a subset of variants cause loss of RET kinase activity and downstream signaling, enzymatic inactivation is not the sole mechanism at play in HSCR.     

Receptor-interacting protein (RIP) kinase family

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research.     

Large differences in proportions of harmful and benign amino acid substitutions between proteins and diseases

Genes and proteins are known to have differences in their sensitivity to alterations. Despite numerous sequencing studies, proportions of harmful and harmless substitutions are not known for proteins and groups of proteins. To address this question, we predicted the outcome for all possible single amino acid substitutions (AASs) in nine representative protein groups by using the PON‐P2 method. The effects on 996 proteins were studied and vast differences were noticed. Proteins in the cancer group harbor the largest proportion of harmful variants (42.1%), whereas the non‐disease group of proteins not known to have a disease association and not involved in the housekeeping functions had the lowest number of harmful variants (4.2%). Differences in the proportions of the harmful and benign variants are wide within each group, but they still show clear differences between the groups. Frequently appearing protein domains show a wide spectrum of variant frequencies, whereas no major protein structural class‐specific differences were noticed. AAS types in the original and variant residues showed distinctive patterns, which are shared by all the protein groups. The observations are relevant for understanding genetic bases of diseases, variation interpretation, and for the development of methods for that purpose.     

Two domains distantly related to protein-tyrosine kinases in the vesicular stomatitis virus polymerase

We have carried out an exhaustive search for amino acid sequence similarities between vesicular stomatitis virus (VSV) proteins and database entries. Unexpectedly, we found that the L polymerase protein contains two blocks of sequence (residues 725-1102 and 1291-1671) with distant but statistically significant similarity to the catalytic domain of tyrosine-specific protein kinases. The first kinase-like region is most similar to members of the Abl subfamily, Fes and Fps (26.6% and 27.3% identity, respectively), whereas the second region is closest to members of the platelet-derived growth factor receptor (PDGFR) subfamily, PDGFR and Kit (30.4% and 25.9% identity, respectively). Multiple alignment of the catalytic domain of these kinases to all three rhabdovirus L protein sequences available (VSV Indiana, VSV New Jersey, and rabies) revealed that the polymerases contain many but not all residues well conserved in the protein kinase family. Similarity was highest for VSV Indiana and lowest for rabies. We conclude that the kinase-like regions in the rhabdoviral L proteins are probably very distantly related to the protein kinase family. The similarities could either reflect contemporary protein kinase activity or represent some other function(s) associated with these large multifunctional polymerase proteins. Our findings also shed new light on questions of the origins and evolution of RNA viruses.     

Molecular cloning of a rac family protein kinase and identification of a serine/threonine protein kinase gene family of Entamoeba histolytica

Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases. These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells. One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene (Eh racl) from an E. histolytica genomic library. The open reading frame of Eh racl was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain. The catalytic domain of Eh racl was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs. Southern blots of ameba DNA showed that the Eh racl gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh racl mRNAs than did nonpathogenic amebae. These studies suggest that E. histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.     

A novel isoform of the B cell tyrosine kinase BTK protects breast cancer cells from apoptosis

Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large‐scale loss‐of‐function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton's tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino‐terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. © 2013 Wiley Periodicals, Inc.     

Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one-third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo.     

wKinMut‐2: Identification and Interpretation of Pathogenic Variants in Human Protein Kinases

Most genomic alterations are tolerated while only a minor fraction disrupts molecular function sufficiently to drive disease. Protein kinases play a central biological function and the functional consequences of their variants are abundantly characterized. However, this heterogeneous information is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut‐2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase‐specific random forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledge bases and the literature is automatically mined, digested, and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic and drug binding are identified. Known protein–protein interactions are reported. Altogether, this information is intended to assist the generation of new working hypothesis to be corroborated with ulterior experimental work. The wKinMut‐2 system, along with a user manual and examples, is freely accessible at http://kinmut2.bioinfo.cnio.es , the code for local installations can be downloaded from https://github.com/Rbbt‐Workflows/KinMut2 .     

SH3-Domain Binding Function of HIV-1 Nef Is Required for Association with a PAK-Related Kinase

HIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex.     

In Silico Prediction of the Effects of Mutations in the Human Mevalonate Kinase Gene: Towards a Predictive Framework for Mevalonate Kinase Deficiency

Mevalonate kinase (MVK) catalyses the phosphorylation of mevalonate. Deficiency of MVK is associated with two rare periodic fever syndromes, mevalonic aciduria (MA), a severe form and hyper‐immunoglobulin‐D syndrome (HIDS), a milder form. An in silico approach was used to analyse the physicochemical and structural effects of 47 disease‐associated variants of MVK. A further 20 variants, which are present in human genome databases, were also analysed. Variants associated with MA are clustered into a “hotspot” consisting of residues 8–35 and 234–338 and tended to result in a prediction of severely reduced protein stability. Four of the uncharacterised variants, p.H24P, p.G198R, p. R253W, and p.G335S, were likely to be associated with MA. This method could be used as the basis for initial predictions of severity when new MVK variants are discovered.     

应激活化蛋白激酶与蛋白激酶p38活性测定

应激活化蛋白激酶（ＳＡＰＫ）和Ｐ＾３２是丝裂素活化蛋白激酶家系（ＭＡＰＫｓ）的主要成员。与应激和某些炎性细胞因子引起的细胞反应有关，参与心血管疾病、细胞凋亡与机体的应激反应。本文主要介绍用免疫沉淀法提取ＳＡＰＫ和ｐ３８，据ＳＡＰＫ和ｐ３８分别可以磷酸化ｃ－Ｊｕｎ和ＡＴＦ－２的原理，采用聚丙烯酰胺电泳（ＳＤＳ－ＰＡＧＥ）方法，分别测定ＳＡＰＫ和ｐ３８将＾３２Ｐ参入其特异性底物的量，反映其活性。     

Genomewide analysis of phytochrome proteins in the phylum Basidiomycota

Phytochromes are photoreceptor proteins involved in the detection of the red and far‐red regions of the visible light spectrum. Fungal phytochromes are hybrid histidine kinases with a conserved domain architecture composed of an N‐terminal photosensory module and a C‐terminal regulatory output module that includes the histidine kinase and response regulator receiver domains. In this study, we have analyzed the distribution, domain architecture, and phylogenetic analysis of phytochrome proteins in 47 published genome sequences among the phylum Basidiomycota. Genome analysis revealed that almost every genome of basidiomycetes contained at least one gene encoding a phytochrome protein. Domain architecture of fungal phytochromes was completely conserved in the identified phytochromes of basidiomycetes, and phylogenetic analysis clustered these proteins into clades related with the phylogenetic classification of this fungal phylum.

     

Targets for Ibrutinib Beyond B Cell Malignancies

Ibrutinib (Imbruvica^?) is an irreversible, potent inhibitor of Bruton's tyrosine kinase (BTK). Over the last few years, ibrutinib has developed from a promising drug candidate to being approved by FDA for the treatment of three B cell malignancies, a truly remarkable feat. Few, if any medicines are monospecific and ibrutinib is no exception; already during ibrutinib's initial characterization, it was found that it could bind also to other kinases. In this review, we discuss the implications of such interactions, which go beyond the selective effect on BTK in B cell malignancies. In certain cases, the outcome of ibrutinib treatment likely results from the combined inhibition of BTK and other kinases, causing additive or synergistic, effects. Conversely, there are also examples when the clinical outcome seems unrelated to inhibition of BTK. Thus, more specifically, adverse effects such as enhanced bleeding or arrhythmias could potentially be explained by different interactions. We also predict that during long‐term treatment bone homoeostasis might be affected due to the inhibition of osteoclasts. Moreover, the binding of ibrutinib to molecular targets other than BTK or effects on cells other than B cell‐derived malignancies could be beneficial and result in new indications for clinical applications.     

Twenty‐One Novel EGFR Kinase Domain variants in Patients with Nonsmall Cell Lung Cancer

Somatic sequence variants in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with nonsmall cell lung cancer (NSCLC). Patients exhibiting sequence variants in this domain that produce kinase activity enhancement, are more likely to benefit from TKIs than patients with EGFR wild‐type disease. Although most NSCLC EGFR‐related alleles are concentrated in a few positions, established protocols recommend sequencing EGFR exons 18–21. In this study, 21 novel somatic variants belonging to such exons in adult Argentinean patients affected with NSCLC are reported. Of these, 18 were single amino acid substitutions (SASs), occurring alone or in combination with another genetic alteration (complex cases), one was a short deletion, one was a short deletion‐short insertion combination, and one was a duplication. New variants and different combinations of previously reported variants were also found. Moreover, two of the reported SASs occurred in previously unreported positions of the EGFR kinase domain. In order to characterize the new sequence variants, physicochemical, sequence and conformational analyses were also performed. A better understanding of sequence variants in NSCLC may facilitate the most appropriate treatment choice for this complex disease.