Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.     

Obtaining a full-length encoding cDNA of Csk family tyrosine kinase from human lymphocytes

Tyrosine kinases of Csk family play important role in the cell growth regulation and normal cell differentiation and also can participate in the process of cancer genesis as oncoproteins. The main function of these tyrosine kinases is the phosphorylation of the Src family tyrosine kinases at their carboxyl terminus, which is the basis of their activity negative regulation. The disturbance of the csk gene expression leads to the increase of the Src tyrosine kinase activity. We have cloned a full-length encoding cDNA of the tyrosine kinase csk gene of human lymphocytes. 1.6-kilobase cDNA encodes the protein, which consists of 12 exons with conserved SH2 and SH3 domains. The homology between this protein and human Csk tyrosine kinase is 99%. A full-length DNA-copy of human lymphocytes RNA can be used for the analysis of csk gene structure in normal and pathologically changed human cells.     

Cloning and analysis of coding cDNA sequence of human leukocyte Csk tyrosine kinase under normal conditions and in choroidal melanoma

The main function of Csk tyrosine kinases is phosphorylation of the C-terminal part of Src tyrosine kinases as a mechanism of their downregulation. A decrease in the expression of csk gene results in the enhancement of Src tyrosine kinase activity. In this study, cDNA containing the full coding sequence of the human leukocyte Csk tyrosine kinase gene has been cloned. The protein encoded by a 1624-bp cDNA fragment has 99% homology to human Csk tyrosine kinase. A comparative sequence analysis of full-length cDNAs for Csk tyrosine kinase of normal lymphocytes and lymphocytes of patients with choroidal melanoma revealed a nucleotide substitution in exon 10 of the gene, which appears to be of diagnostic significance. It has been shown that the risk of choroidal melanoma correlated with the frequency of this allele.     

Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina

Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.     

A novel tyrosine kinase, hyk, expressed in murine embryonic stem cells.

To identify tyrosine kinases which play roles in mammalian early development, the 3' rapid amplification of cDNA ends (RACE) was performed on mouse embryonic stem (ES) cells. Among eight tyrosine kinases thus identified, we report here a novel tyrosine kinase, hyk (adhesion structures linked tyrosine kinase). The sequences of the 4.7 kb cDNA indicated the presence of RGD motif and three epidermal growth factor-like domains put between two immunoglobulin-like domains and three fibronectin type III domains in its extracellular region. It is strongly expressed in ES cells and later stages of embryos, but at low levels in midgestation embryos. It is also expressed at a low level in neural precursor cells from 10-day embryos, but at high levels in embryonic day 15 and neonatal brains. In adult tissues it is expressed ubiquitously.     

Comparative proteome bioinformatics: identification of a whole complement of putative protein tyrosine kinases in the model flowering plant Arabidopsis thaliana

Phosphorylation by protein tyrosine kinases is crucial to the control of growth and development of multicellular eukaryotes, including humans, and it also seems to play an important role in multicellular prokaryotes. A plant tyrosine-specific kinase has not been identified yet; hence, plants have been suggested to share with unicellular eukaryote yeast a tyrosine phosphorylation system where a limited number of stress proteins are tyrosyl-phosphorylated only by a few dual-specificity (serine/threonine and tyrosine) kinases. However, preliminary evidence obtained so far suggests that tyrosine phosphorylation in plants depends on the developmental conditions. Since sequencing of the genome of the model flowering plant Arabidopsis thaliana has been recently completed, we have performed a bioinformatic screening of the whole Arabidopsis proteome to identify a model complement of bona fide protein tyrosine kinases. In silico analyses suggest that < 4% of Arabidopsis kinases are tyrosine-specific kinases, whose gene expression has been assessed by a preliminary polymerase chain reaction screening of an Arabidopsis cDNA library. Finally, immunological evidence confirms that the number of Arabidopsis proteins specifically phosphorylated on tyrosine residues is much higher than in yeast.     

Differential display of protein tyrosine kinase cDNAs from human fetal and adult brains

Here we describe a differential display method for surveying the expression of most protein tyrosine kinases and applying it to cDNAs from human fetal and adult brains. The method involves two selective steps for processing the mRNA. At each step, degenerate oligonucleotide primers derived from highly conserved regions of the catalytic domain of the kinases are used. In the display with BstYI and BsiHKI digests of the cDNA, 65% and 59% of a total of 72 and 63 bands, respectively, represented fragments from a total of 27 different tyrosine kinases. The expression levels of the kinases in the display were comparable with those measured by RT-PCR. This method offers a relatively specific way to display differentially expressed gene families in any tissue and cell type.     

Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum.

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.     

Differential association of cellular proteins with family protein-tyrosine kinases

We have sought to identify candidate substrates for src family protein-tyrosine kinases potentially important for transformation. Transfected NIH/3T3 cells, each overexpressing a normal or activated version of the fyn, fgr, or src translational product, were examined using antibody to phosphotyrosine as a probe. Expression of each cDNA induced similar but distinct patterns of tyrosine phosphorylated cellular proteins, with the extent of phosphorylation being greatest in cells expressing an activated kinase. A 70-kDa tyrosine-phosphorylated protein was found to associate with the activated fyn gene product. A protein designated p130, tyrosine phosphorylated in vitro, and in vivo, was found to physically associate with the activated product of each src family gene examined. Physical interaction of three different highly transforming tyrosine kinases with a common cellular protein suggests that p130 may play an important role in transformation induced by src family kinases.     

Characterization of elk, a brain-specific receptor tyrosine kinase.

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.     

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.     

Identification of immune-related protein kinases from mosquitoes (Aedes aegypti)

Protein kinases are known to be involved in signal transduction for numerous physiological events. However, little is known about the roles of protein kinases in insect immunity. A fragment around 150 bp was amplified by polymerase chain reaction using cDNA templates from bacterial inoculated mosquitoes and primers corresponding to the conserved domain of protein kinases. Based on sequence analysis, 11 groups of protein kinases were characterized including 3 nonreceptor tyrosine kinases, 3 receptor tyrosine kinases, 3 serine/threonine kinases, and 2 novel protein kinases. The most abundant kinase obtained in this study reveals a high degree of similarity to human cholinesterase-related cell division controller (CHED) protein kinase. The expression of this mosquito CHED-like kinase is not detectable in normal female mosquitoes, but induced only after bacterial inoculation and trauma. A mosquito protein kinase was demonstrated to share homology with a plant Tousled gene, but has not yet been characterized in the animal system. In addition, analysis of the sequences of several protein kinases cloned from mosquitoes suggests that they might be involved in the regulation of cellular or humoral immunity.     

Assignment of tyrosine-specific T-cell phosphatase to conserved syntenic groups on human chromosome 18 and mouse chromosome 18.

Phosphorylation of proteins on tyrosine is crucially involved in signal transduction and mitogenesis and is regulated by both kinases and phosphatases. Recently, a number of soluble and transmembrane receptor-linked protein tyrosine phosphatases (PTPase) have been characterized. Among these is a 48.4-kDa PTPase encoded by a cDNA isolated from a T-lymphocyte library by low-stringency screening with probes derived from placental PTPase 1B. A human T-cell PTPase (PTPT) cDNA and somatic cell hybrids were used to assign a PTPT gene to conserved syntentic groups on human chromosome 18 and on mouse chromosome 18. Two unlinked sequences, one on human chromosome 1, were also detected.     

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.     

Rapid detection of protein tyrosine kinase activity in recombinant yeast expressing a universal substrate

Yeast two-hybrid systems are powerful proteomics tools for the discovery of protein-protein interactions. However, these systems are typically unable to detect interactions dependent on post-translational modifications such as tyrosine phosphorylation. We report a novel yeast tribrid system that expresses a potentially universal protein tyrosine kinase (PTK) substrate to detect diverse PTKs. Validation with the oncogenic kinases v-Abl and v-Src, which exhibit divergent substrate specificities, demonstrated significant potential for cloning PTKs en masse from cDNA libraries.     

Isolation and characterization of a human putative receptor protein kinase cDNA STYK1*

Protein kinases (PKs) represent a well studied but most diverse protein superfamily. The covalent, reversible linkage of phosphate to serine, threonine, and tyrosine residues of substrate proteins by protein kinases is probably ubiquitous cellular mechanism for regulation of physiological processes. It is known to us that most signaling pathways impinge at some point on protein kinases. Here we report a human putative receptor protein kinase cDNA STYK1. The STYK1 cDNA is 2749 base pairs in length and contains an open reading frame encoding 422 amino acids. The STYK1 gene is mapped to human chromosome 12p13 and 11 exons were found. RT-PCR showed that STYK1 is widely expressed in human tissues.