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G Superti-Furga

Publications and source records attributed to G Superti-Furga.

At least 37 records · Page 2Linked to original sources
PubMed1997

A functional screen for regulators of the c-Abl protein tyrosine kinase.

c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells. Several mutations can activate Abl and convert it into an oncogene. In man, chromosomal translocations result in fusion proteins associated with chronic myelogenous leukemias and some acute lymphocytic leukemias. In viral forms of abl, gag sequences are fused to Abl portions resulting in a deletion of N-terminal sequences. To study c-Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion. c-Abl causes growth arrest followed by death of the cells. Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme. An SH3 domain deletion mutant of Abl is as active as c-Abl in yeast indicating that there is no intrinsic regulation of c-Abl occurring via the SH3 domain and suggesting that the inhibitory effect of the SH3 domain observed in cells of vertebrate origin is mediated by a factor that is absent in fission yeast. We have used this assay to functionally screen a human cDNA library for molecules able to counteract the lethal effect of c-Abl expression. We are currently in the process of characterising the isolated clones. We hope to identify among them the molecule(s) responsible for regulating c-Abl activity in human cells.

Animals↗
PubMed1996

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.

Amino Acid Sequence↗
PubMed1996

Analysis of human c-Abl tyrosine kinase activity and regulation in S. pombe.

c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells. Several mutations, including deletions of the SH3 domain, can activate abl and convert it into an oncogene. To study c-Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion. c-Abl, but not a kinase inactive form of the molecule, causes growth arrest followed by death of the cells. Concomitant to Abl expression we observed extensive phosphorylation of endogenous proteins on tyrosine. Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme. An SH3 domain deletion mutant of Abl is equally active as wild type c-Abl in yeast, even under conditions allowing detection of subtle differences. These results demonstrate that there is no intrinsic regulation of c-Abl kinase activity via the SH3 domain and suggest that the inhibitory effect of the SH3 domain observed in mammalian cells is medicated by a factor that is absent in fission yeast. Expression of Ab1 S.pombe provides a novel quantitative assay for ab1 activity and regulation.

Gene Expression Regulation, Enzymologic↗
PubMed1996

A functional screen in yeast for regulators and antagonizers of heterologous protein tyrosine kinases.

Tyrosine phosphorylation exerts a pivotal role in cell regulation processes of higher eukaryotes. Tight control of the activity of protein tyrosine kinases is crucial for ordered phosphorylation to occur. We have developed a functional screen for tyrosine kinase regulators using c-Src, the first cellular protein tyrosine kinase described, as a prototype; and fission yeast, Schizosaccharomyces pombe, as a genetically amenable host system. Inducible expression of c-Src in fission yeast is lethal. We have screened human cDNA libraries for clones able to counteract the lethal effect of Src. Two different classes of cDNAs, which we called SAS for sequences antagonizing Src, were obtained. The first class encodes for the protein tyrosine kinase Csk, known to regulate Src activity through phosphorylation of the C-terminal tyrosine. The second class consists of clones encoding three different tyrosine phosphatases, counteracting Src action by dephosphorylation of Src substrates and by dephosphorylation of Src itself. The system described here can be applied to identify regulators of other heterologous tyrosine kinases, including receptor-type tyrosine kinases, which impair growth of S. pombe.

Biotechnology↗
PubMed1996

The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.

Amino Acid Sequence↗
PubMed1995

Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk.

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule. A different protein tyrosine kinase, Csk, is largely responsible for this regulation. The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain. Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed. Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein. Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src. Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins. Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain. The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.

CSK Tyrosine-Protein Kinase↗
PubMed1995

Regulation of the Src protein tyrosine kinase.

Members of the Src family of protein tyrosine kinases are involved in a variety of cellular processes, including cell growth, cell differentiation and neuronal signalling. N-terminal to the catalytic domain, Src family members contain a Src homology 2 (SH2) domain, a Src homology 3 (SH3) domain, and a unique domain, all capable of protein-protein interactions. Negative regulation by phosphorylation of a conserved tyrosine residue at the C-terminal tail of the molecules is characteristic of this family of enzymes. Phosphorylation of this residue causes the intramolecular interactions of the SH2 domain with the tail, and of the SH3 domain with an as yet undefined region, probably within the catalytic domain. Enzymatically active Src family kinases, on the other hand, are phosphorylated at a tyrosine in the middle of the catalytic domain and phosphorylation of this residue is a prerequisite for high activity. Regulators of these enzymes may thus act by altering the phosphorylation state of the two key tyrosine residues or by interfering with the regulatory intramolecular interactions, either by direct binding or by modification of the interfaces involved.

Gene Expression Regulation, Enzymologic↗
PubMed1995

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.

3T3 Cells↗
PubMed1995

Structure-function relationships in Src family and related protein tyrosine kinases.

There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains.

Animals↗
PubMed1993

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.

Amino Acid Sequence↗
PubMed1993

The Src family of protein tyrosine kinases: regulation and functions.

Most of the nine members of the Src family of tyrosine kinases are restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more ubiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to PDGF, and we therefore conclude that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain: phosphorylated tail interaction repressing Src activity. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Identification of Fos target genes by the use of selective induction systems.

c-Fos is a major component of the transcription factor AP-1 which has been implicated in the control of cell proliferation and differentiation as well as in transformation. In order to identify Fos target genes involved in these processes, we have taken advantage of the regulatory properties of the hormone-binding domain of the human estrogen receptor to develop transcriptional and post-translational induction systems, both of which allow selective elevation of Fos activity within a cell. Using this approach we have searched for Fos-responsive genes in rat fibroblasts and PC12 cells. Here we describe the identification and regulation of five Fos-responsive genes encoding a transcription factor (Fra-1), a secreted protein (Fit-1), a biosynthetic enzyme (ODC) and two membrane-associated proteins (annexin II and V), respectively. The post-translational induction system was also used to study the Fos-mediated block of neuronal differentiation of PC12 cells. These experiments demonstrate that Fos activity is dominant over NGF function and interferes with the expression of late NGF-inducible genes.

Animals↗
PubMed1991

Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins.

The protooncogene c-fos has been implicated in the control of proliferation and transformation of fibroblasts, and its protein product is an essential component of transcription factor AP1. The important target genes and, hence, the molecular mechanism of Fos function are, however, still unknown, partly due to the lack of a tightly regulated Fos-induction system. Here we show that different activities of the Fos protein can be controlled hormonally by fusing the mouse c-Fos protein to the ligand-binding domain of either the rat glucocorticoid or the human estrogen receptor. These fusion proteins stimulate AP1-dependent transcription and repress endogenous fos mRNA synthesis in a strictly hormone-dependent manner. Expression of these chimeric proteins in rat fibroblasts results in fast, reversible, and tightly controlled transformation in response to hormone. A Fos-estrogen receptor expressing cell line was used to isolate Fos-responsive genes by subtractive cDNA cloning. Run-on analysis of one of these genes showed that its transcription is rapidly and directly regulated by the hormone-activated Fos-estrogen receptor protein, demonstrating the potential of this induction system for identifying Fos target genes.

Animals↗
PubMed1989

The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein.

Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH.

Base Sequence↗
PubMed1989

The protein CDP, but not CP1, footprints on the CCAAT region of the gamma-globin gene in unfractionated B-cell extracts.

We have identified, by DNase I footprinting, six different factors that interact with the promoter of the human A gamma-globin gene in nuclear extracts of the B-cell line BJA-B. Among them is the vertebrate homologue of the sea-urchin CCAAT displacement protein (CDP) which footprints over the entire duplicated CCAAT region. The CCAAT-binding factor CP1, a potential activator of the gamma-globin promoter, is able to bind to its proximal recognition sequence only once it has been partially enriched and separated from CDP. The factor CDP has an apparent molecular mass of 200 kDa and differs from CP1 by its footprint pattern and competition behavior.

B-Lymphocytes↗
PubMed1989

Developmental and tissue-specific regulation of a novel transcription factor of the sea urchin.

We have identified a novel transcription factor that interacts with the promoter of four tissue-specific late histone H2A-2 and H2B-2 genes of the sea urchin by DNase I footprint, mobility shift, and methylation interference analyses. The binding site for this factor is required for efficient transcription of the H2B-2.1 gene both in vitro in nuclear extracts of gastrula embryos and in vivo in microinjected sea urchin embryos. This factor binds with equal affinity to the recognition sequences of all four histone genes in cross-competition assays. Moreover, the binding site of the H2B-2.2 promoter can functionally substitute for that of the H2B-2.1 gene in in vivo expression experiments. Nevertheless, all four binding sites share little sequence homology with each other. This transcription factor increases in abundance during embryogenesis and has been detected in the adult sea urchin only in the tube feet, where the late H2A-2 and H2B-2 genes are expressed specifically. Therefore, we refer to this factor as tissue-specific activator protein (TSAP). The close correlation between the presence of TSAP and the expression pattern of the late H2A-2 and H2B-2 genes suggests that this transcription factor is directly responsible for the developmental and tissue-specific regulation of these genes.

Aging↗