[Regulation of Src family tyrosine-kinases by Csk].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S Nada
Publications and source records attributed to S Nada.
Non-receptor tyrosine kinases in mammalian neurogenesis.
Several members of the Src family of non-receptor tyrosine kinases are expressed at high levels in embryonic neural tissues as well as in adult brain. Relatively little has been known, however, about their roles in neural development. Attempts to clarify this by production of mutant mice have been unsuccessful because of gene redundancy. We earlier isolated a new cytoplasmic protein tyrosine kinase, Csk, and showed that it inactivates uniquely all members of non-receptor tyrosine kinases in vitro. Here, we have generated Csk-deficient mouse embryos and shown that Csk is indeed an indispensable negative regulator for all non-receptor tyrosine kinases in vivo, and that regulated activity of these kinases is essential for normal development of mice at the neural stage. The signaling pathway through Src-family kinases during neurulation is also discussed.
Amino Acid Sequence↗
Functional and physical interaction of protein-tyrosine kinases Fyn and Csk in the T-cell signaling system.
The Src-like protein-tyrosine kinase Fyn is associated with T-cell antigen receptor. Transient expression of actively mutated Fyn, having Phe-528 instead of Tyr-528 or Thr-338 instead of Ile-338, in Jurkat T-cells stimulated the serum response element (SRE), 12-O-tetradecanoyl-phorbol-13-acetate response element, cyclic AMP response element, and c-fos promoter. The stimulation of SRE was particularly prominent not only with active Fyn but also with normal (wild-type) Fyn. SRE was also stimulated by both normal and active Lck. Furthermore, normal and active Fyn stimulated transcription from the IL-2 gene promoter when transfected cells were stimulated by concanavalin A plus 12-O-tetradecanoylphorbol-13-acetate. Under the same conditions, Lck did not stimulate IL-2 promoter unless it was activated by mutation. Interestingly, a mutant Fyn, which has deletions within the SH2 region and so is able to transform chicken embryo fibroblasts, did not stimulate either the c-fos or IL-2 promoter, suggesting the importance of this region in T-cell signaling. Csk, which phosphorylates tyrosine residues in the negative regulatory sites of Src family kinases, down-regulated Fyn- and Lck-mediated stimulation of the serum response element and Fyn-mediated enhancement of IL-2 promoter activity. These data suggest that Fyn and Lck, whose activities are regulated by Csk, are involved in different phases of T-cell activation.
Amino Acid Sequence↗
Constitutive activation of Src family kinases in mouse embryos that lack Csk.
Csk is a novel cytoplasmic protein-tyrosine kinase that has been shown to inactivate members of the Src family of protein-tyrosine kinases in vitro. To examine the function of Csk in vivo, Csk-deficient mouse embryos were generated by gene targeting in embryonic stem cells. These embryos were developmentally arrested at the 10 to 12 somite stage and exhibited growth retardation and necrosis in the neural tissues. The kinase activity of p60c-src, p59fyn, and p53/56lyn in these embryos was greatly enhanced as an apparent consequence of enhanced specific activity. The increase in kinase activity was associated with an increase in tyrosine phosphorylation of several proteins, especially those around 85 and 120 kd. Thus, these results suggest that Csk indeed acts as an indispensable negative regulator of Src family kinases in vivo.
Animals↗
Primary structure of the gene for glycyl-tRNA synthetase from Bombyx mori.
The primary structure of the gene encoding Bombyx mori glycyl-tRNA synthetase was determined by sequence analysis of one cDNA and two genomic clones. The sequence of the protein deduced from the nucleotide sequence was verified by sequence analysis of eight peptides. The M(r) 77,667 protein is encoded in a single open reading frame of 2061 nucleotides. There are no introns in the gene. The deduced protein sequence has no obvious similarity to Escherichia coli glycyl-tRNA synthetase but contains a sequence in its amino terminus that is similar to a sequence found in the Drosophila melanogaster and human glutamyl-tRNA synthetases, the hamster and human histidyl-tRNA synthetases, bovine tryptophanyl-tRNA synthetase, and the mammalian peptide chain release factor. The B. mori glycyl-tRNA synthetase also has sequence similarity with the Saccharomyces cerevisiae (cytoplasmic and mitochondrial), E. coli, and human threonyl-tRNA synthetases. This sequence similarity occurs in a sequence motif that is characteristic of other class II aminoacyl-tRNA synthetases. Two transcription start sites approximately 100 nucleotides apart were identified by ribonuclease mapping. One of the transcription start sites is used preferentially in the posterior silk gland. The peak in mRNA accumulation occurs 80-100 h prior to the peak in glycyl-tRNA synthetase activity and enzyme protein.
Amino Acid Sequence↗
A novel ES cell line, TT2, with high germline-differentiating potency.
In producing mutant mice by gene-targeting and gene-trapping in embryonic stem (ES) cells, the efficient colonization of the mutant ES cells into germline is still a critical matter. We have established a new line of ES cells, TT2, from an F1 embryo between a C57BL/6 female and a CBA male. When the TT2 cells were injected into blastocysts, the colonization into each tissue was very low. However, when injected into eight-cell embryos, the cells segregated inside the blastomeres, localized in an inner cell mass of blastocysts developed 1 day later, and colonized efficiently in each tissue of the pups. The pups were disproportionately male, about half of which were composed of TT2-derived cells primarily; in more than 70% of the males, TT2-derived cells were dominant, accounting for over half of the total cells. When these males were mated, they exclusively yielded TT2-derived offspring. The germline-differentiating potency was stable during 3 weeks of culture. Twenty-one of 24 mutant clones independently isolated yielded germline chimeras, and 19 clones yielded them in a rate comparable to that of the parent cells. Thus, TT2 cells can serve as a valuable vehicle for the production of mutant mice.
Animals↗
A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene.
In producing mutant mice by gene targeting in embryonic stem (ES) cells, the efficient isolation of the homologous recombinants is still a critical step. We previously reported on a negative selection using the diphtheria toxin A (DT-A) fragment gene for homologous recombinants (1). It was efficient but limited to gene loci expressed in ES cells. For wider applicability of this negative selection to many gene loci not expressed or expressed at low levels in ES cells, we exploited a novel targeting vector composed of a polyA-less neo gene, a mRNA destabilizing signal, a pausing signal for RNA polymerase II from the minute virus of mice, and the DT-A gene. There was about a 30-fold decrease in frequency of G418-resistant colonies with this strategy against that using only the neo gene in the vector, and homologous recombinants were obtained at frequencies of more than 1/50 among G418 resistant cells at fyn, csk, c-mos, and insulin receptor substrate-1 gene loci.
Animals↗
Phosphorylation of Src mutants at Tyr 527 in fibroblasts does not correlate with in vitro phosphorylation by CSK.
In normal fibroblasts, the product of the cellular src gene, p60c-src or Src, is repressed by phosphorylation at its C-terminal tyrosine residue, Tyr 527. Mutations in Src that prevent phosphorylation cause enzymatic activation and malignant transformation. The tyrosine kinases that phosphorylate Src at Tyr 527 in vivo have not been identified, but a tyrosine kinase known as CSK is an excellent candidate. CSK has the unusual ability to phosphorylate Src in vitro only at Tyr 527. To examine whether CSK has the appropriate sequence specificy to explain the phosphorylation of Src at Tyr 527 in fibroblasts, we have made use of a set of C-terminal substitution mutants of Src. These mutants were previously characterized for their levels of Tyr 527 phosphorylation when expressed in Rat2 fibroblasts. The ability of CSK to phosphorylate selected mutants has now been tested, using both in vitro phosphorylation assays and co-expression of CSK with the Src mutants in a heterologous organism, Saccharomyces cerevisiae. We also tested whether the mutant Src molecules could autophosphorylate at Try 527, by examining the phosphorylation state of catalytically active forms expressed in the absence of CSK in yeast cells. The results show that CSK has strict sequence specificity for the normal Src sequence, although it can also phosphorylate the Lck sequence. The other mutant Src molecules tested were not phophorylated by CSK, even though some of these mutants are highly phosphorylated at Tyr 527 in Rat 2 cells. All the mutants that are phosphorylated at Tyr 527 in Rat2 cells are also able to autophosphorylate at Tyr 527. The results suggest that CSK, autophosphorylation, and phosphorylation by kinases other than CSK, may all contribution to repressing Src catalytic activity in fibroblasts.
Amino Acid Sequence↗
Molecular cloning and expression of chicken C-terminal Src kinase: lack of stable association with c-Src protein.
Cloning and sequencing of chicken C-terminal Src kinase (CSK), a tyrosine kinase that phosphorylates the regulatory C-terminal tyrosine residue present on cytoplasmic tyrosine kinases of the Src family, demonstrated a high degree of interspecies conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of autophosphorylation sites distinguishes CSK from other tyrosine kinases. CSK is unique and does not belong to a gene family, suggesting that it may phosphorylate other members of the Src family of tyrosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regulatory step in the control of c-Src kinase activity, such an association was investigated by immunoprecipitation and Western blotting as well as intracellular localization studies. Although some portions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src.
Amino Acid Sequence↗
CSK: a protein-tyrosine kinase involved in regulation of src family kinases.
The functions of src family protein-tyrosine kinases are thought to be regulated negatively by the phosphorylation of highly conserved tyrosine residues close to their carboxyl termini. Recently we have purified and cloned a protein-tyrosine kinase (designated as CSK) that can specifically phosphorylate the negative regulatory site of p60c-src. To elucidate the relationship between CSK and other types of src family kinases, we investigated the tissue distribution of CSK and examined whether CSK could phosphorylate the negative regulatory sites of src family kinases other than p60c-src. Western blot analysis indicated that CSK was enriched at the highest level in lymphoid tissues in which the expression of p60c-src is considerably lower than those of other types of src family kinases. CSK phosphorylated p56lyn and p59fyn, which are known to be expressed in lymphoid tissues at a relatively high level. The putative regulatory site, tyrosine 508, was found to be essential for phosphorylation in p56lyn, and the kinase activities of these src family kinases were repressed by phosphorylation with CSK. These findings raise the possibility that CSK might act as a universal regulator for src family kinases.
Amino Acid Sequence↗
Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.
The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.
Amino Acid Sequence↗