RESUMO
We have cloned a member of the STE20/SPS1 protein kinase family from a transformed rat pancreatic beta cell line. SPAK (STE20/SPS1-related, proline alanine-rich kinase) belongs to the SPS1 subfamily of STE20 kinases and is highly conserved between species. SPAK is expressed ubiquitously, although preferentially in brain and pancreas. Biochemical characterization of SPAK catalytic activity demonstrates that is a serine/threonine kinase that can phosphorylate itself and an exogenous substrate in vitro. SPAK is immunoprecipitated from transfected mammalian cells as a complex with another, as yet uncharacterized, serine/threonine kinase which is capable of phosphorylating catalytically-inactive SPAK and myelin basic protein in an in vitro kinase assay. SPAK specifically activates the p38 pathway in cotransfection assays. Like MST1 and MST2, SPAK contains a putative caspase cleavage site at the junction of the catalytic domain and the C-terminal region. Full-length SPAK is expressed in the cytoplasm in transfected cells, while a mutant corresponding to caspase-cleaved SPAK is expressed predominantly in the nucleus. The similarity of SPAK to other SPS1 family members, its ability to activate the p38 pathway, in addition to its putative caspase cleavage site, provide evidence that SPAK may act as a novel mediator of stress-activated signals. Oncogene (2000) 19, 4290 - 4297
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/enzimologia , Linhagem Celular Transformada , Núcleo Celular/enzimologia , Citoplasma/enzimologia , DNA Complementar/genética , Indução Enzimática , Genes , Humanos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/patologia , Dados de Sequência Molecular , Família Multigênica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/fisiologia , Especificidade de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estresse Fisiológico/enzimologia , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Divisão Celular/genética , Sobrevivência Celular/genética , Ativação Enzimática/genética , Receptores ErbB/genética , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Proteína Adaptadora GRB2 , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Mitógenos/farmacologia , Mutação/genética , Fosforilação , Fosfotirosina/análise , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
The Epidermal Growth Factor (EGF) receptor appears to require a fully active tyrosine kinase domain to transmit mitogenic signals. However, waved-2 mice carrying a mutation in the alpha-helix C of their EGF-R, which abolishes tyrosine kinase activity, only display a mild phenotype and are fully viable. This suggests that the mutant EGF-R signals through heterodimerization with endogenous, kinase active members of the EGF-R family such as ErbB-2 or ErbB-4. We have examined the biochemistry of EGF-Rs carrying mutations in the alpha-helix C of the human EGF-R (V741G and Y740F), in the ATP binding site (K721R) and at the C-terminus (CT957), by expression in BaF/3 cells which are devoid of EGF-R family members. The in vitro kinase activity of the alpha-helix C EGF-R mutants was severely impaired as a result of reduced phosphotransfer activity without appreciable changes in the affinity for either ATP or peptide substrate. Surprisingly, EGF stimulation of cells carrying the different mutant or wild type EGF-Rs resulted in tyrosine phosphorylation of EGF-R proteins; this phosphorylation was abolished in crude plasma membrane preparations, and appears to be due to activation of a membrane-associated or a cytosolic kinase. Receptor-mediated internalization of EGF was profoundly suppressed in the V741G, K721R and CT957 receptor mutant, and high affinity EGF binding was undetectable in the V741G and K721R receptors. We conclude that specific residues in the C-helix of the EGF-R kinase are essential for full kinase activity; mutations in this region do not affect ATP binding, but impair the receptors' phosphotransfer ability. High affinity binding of EGF is not dependent on tyrosine kinase activity or sequences in the C-terminus.
Assuntos
Receptores ErbB/genética , Receptores ErbB/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Animais , Linfócitos B , Sítios de Ligação , Membrana Celular/metabolismo , Endocitose , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/química , Citometria de Fluxo , Humanos , Cinética , Ligantes , Camundongos , Modelos Moleculares , Mutação , Fosforilação , Fosfotirosina/metabolismo , Estrutura Secundária de ProteínaRESUMO
PTPL1 is an intracellular protein-tyrosine phosphatase that contains five PDZ domains. Here, we present the cloning of a novel 150-kDa protein, the four most C-terminal amino acid residues of which specifically interact with the fourth PDZ domain of PTPL1. The molecule contains a GTPase-activating protein (GAP) domain, a cysteine-rich, putative Zn2+- and diacylglycerol-binding domain, and a region of sequence homology to the product of the Caenorhabditis elegans gene ZK669.1a. The GAP domain is active on Rho, Rac, and Cdc42 in vitro but with a clear preference for Rho; we refer to the molecule as PTPL1-associated RhoGAP 1, PARG1. Rho is inactivated by GAPs, and protein-tyrosine phosphorylation has been implicated in Rho signaling. Therefore, a complex between PTPL1 and PARG1 may function as a powerful negative regulator of Rho signaling, acting both on Rho itself and on tyrosine phosphorylated components in the Rho signal transduction pathway.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar , Ativação Enzimática , Proteínas Ativadoras de GTPase , Dados de Sequência Molecular , Ligação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The intracellular protein-tyrosine phosphatase PTPL1 has five PDZ domains and one of them, PDZ 2, has previously been shown to interact with the C-terminal tail of Fas, a member of the tumor necrosis factor receptor family. Using a peptide binding assay, we show that not only PDZ 2 but also PDZ 4 of PTPL1 interacts with high affinity with peptides derived from the C terminus of Fas. The five most C-terminal amino acid residues of Fas influence the affinity of the interaction. Whereas the glutamine and isoleucine residues in the 4th and 5th positions from the C terminus affect the interaction in a negative and positive manner, respectively, the three C-terminal amino acid residues (SLV) are necessary and sufficient for a high affinity interaction to occur. Both the carboxyl group and side chain of the valine residue at the C terminus of Fas are essential, and the leucine and serine residues in the 2nd and 3rd positions, respectively, from the C terminus are important for the interactions with PDZ 2 and PDZ 4 of PTPL1.
Assuntos
Proteínas Tirosina Fosfatases/química , Receptor fas/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Proteínas Recombinantes de Fusão , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
Changes in mRNA expression are physiological regulatory mechanisms and frequently deliver important information regarding functions of corresponding gene products. We investigated changes of abundantly expressed mRNAs of two transmembrane protein tyrosine phosphatases, LRP (leukocyte common antigen-related phosphatase) and mRPTP-sigma. The LRP mRNA expression was modulated by platelet derived growth factor (PDGF) treatment and seems to be regulated by PDGF receptor kinase. The expression of mRPTP-sigma mRNA was low in actively cycling cells, like those in the exponential phase of growth or those treated with different growth factors. In cells whose growth was arrested by contact inhibition at high cell density or by serum starvation at low cell density mRPTP-sigma mRNA level increased. The possible implications of these mRNA expression patterns are discussed.
Assuntos
Substâncias de Crescimento/farmacologia , Proteínas Tirosina Fosfatases/genética , Receptores de Superfície Celular , Células 3T3 , Animais , Divisão Celular , Transformação Celular Viral , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Camundongos , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
A novel cytoplasmic protein tyrosine phosphatase (PTP), PTPL1, was identified and cloned using a polymerase chain reaction-based approach. Overlapping cDNA clones encompass an open reading frame of 7398 base pairs, predicting a protein of 2466 amino acid residues with a molecular mass of 275 kDa. PTPL1 has a wide tissue distribution, a 9.5-kilobase transcript being expressed in most tissues. Peptide antisera against PTPL1 specifically precipitate a protein with an apparent mass of 250 kDa. PTPL1 has a PTP domain located in the COOH terminus, and the protein was shown to dephosphorylate 32P-labeled myelin basic protein. In the non-enzymatic part of PTPL1, three different structural motifs can be identified. Two of these are often found in proteins at the interface between the plasma membrane and the cytoskeleton, i.e. a 300-amino acid domain with similarity to the band 4.1 superfamily, and a region consisting of five GLGF repeats, an 80-amino acid repeat found in a variety of cytoskeleton-associated proteins. In addition to these structures PTPL1 has a region that fulfills the criteria for a leucine zipper motif. PTPL1 is the hitherto largest PTP identified and the only one known which contains a leucine zipper motif.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas Tirosina Fosfatases/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Proteínas do Citoesqueleto/metabolismo , DNA Complementar , Humanos , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Proteína Tirosina Fosfatase não Receptora Tipo 13 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is a 90 kDa protein consisting of two non-covalently associated subunits. In addition to the previously identified 1.8 kilobase (kb) PD-ECGF/TP mRNA, the human epidermoid carcinoma cell line A431 was found to express 3.0 kb and 3.2 kb transcripts. cDNA cloning of the larger transcripts revealed that they contain long 5' leader sequences of 1.5 kb and 1.7 kb, respectively, and the same open reading frame encoding PD-ECGF/TP as that of the 1.8 kb transcript. Comparison with the published PD-ECGF/TP genomic sequence shows that the long 5' leader sequence contain 7 of 8 copies of Sp-1 binding sites present in the transcription promoter region of the 1.8 kb transcript.
Assuntos
Timidina Fosforilase/genética , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Complementar/análise , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
We have previously identified and purified transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes (Ichijo, H., Rönnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., and Miyazono, K. (1991) J. Biol. Chem. 266, 22459-22464). One of these TGF-beta 1-binding proteins, with a molecular weight of 40,000, was purified to homogeneity and subjected to amino acid sequence analysis. The amino acid sequences obtained were used to isolate two closely related cDNA clones from a porcine uterus cDNA library. The deduced amino acid sequences revealed that both cDNAs encoded proteins that were mainly composed of fibrinogen-like and collagen-like domains. Therefore, they were denoted ficolin-alpha and ficolin-beta. Expression of ficolin-alpha and -beta cDNA in mammalian cells revealed that ficolin forms dimers, trimers, and several higher order of oligomers, whose molecular weights fit well with those of the purified TGF-beta 1-binding proteins from porcine uterus. Moreover, immunoblotting analysis using a peptide anti-serum against ficolin indicated that the TGF-beta 1-binding proteins identified in porcine uterus are ficolin-alpha, -beta, and their oligomers or closely related molecules. However, recombinant ficolin-alpha and -beta did not bind TGF-beta 1, despite the similarities in molecular weights and immunoreactivity with the material from the natural source. It is possible that a specific posttranslational modification of ficolin or interaction with another component is needed for TGF-beta 1 binding. Analysis by Northern blotting revealed that the expression of ficolin-alpha mRNA is relatively restricted and most abundant in placenta and lung. On the other hand, ficolin-beta was mainly expressed in skeletal muscle. The in vivo functions of ficolin will be discussed.
Assuntos
Proteínas de Transporte/genética , Colágeno/genética , Fibrinogênio/genética , Lectinas , Útero/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular/métodos , DNA , Feminino , Expressão Gênica , Biblioteca Gênica , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Suínos , Transfecção , Fator de Crescimento Transformador beta/metabolismo , FicolinasRESUMO
We describe the production and characterization of a mouse mAb, S-450-33.2, recognizing the Ly-9.2 specificity. This mAb was used to purify Ly-9 molecules from lymphoid cell lines, and the amino-terminal amino acids were determined. The mAb was also used in a eukaryotic expression system, to isolate cDNA clones encoding Ly-9. Analysis of RNA showed that Ly-9 expression is lymphocyte specific, as determined by the presence of a single hybridizing 2.4-kb species found only in lymphoid cells. Genomic DNA analysis indicated that Ly-9 is encoded by a single-copy gene of 10 to 15 kb. The predicted polypeptide belongs to the Ig superfamily of cell surface molecules with four extracellular Ig-like domains, i.e., a non-disulfide-bonded V domain, a truncated C2 domain with two disulfide bonds, a second non-disulfide-bonded V domain, and a truncated C2 domain with two disulfide bonds (V-C2-V-C2). The sequence data also support the view that Ly-9 belongs to the subgroup of the Ig superfamily that includes Bcm-1, CD2, and LFA-3.
Assuntos
Antígenos Ly/genética , DNA/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Antígenos Ly/análise , Antígenos Ly/química , Sequência de Bases , Northern Blotting , Southern Blotting , DNA/química , Genes de Imunoglobulinas , Camundongos , Dados de Sequência Molecular , TransfecçãoRESUMO
Using tumour cell lines expressing specific isoforms of murine Ly-5 (molecular weights of 180,000, 200,000 and 240,000) we find that all forms of Ly-5 and immuno-affinity purified forms of Ly-5 contain tyrosyl phosphatase activity. These results demonstrate that these isoforms of Ly-5 belong to the same family of functional receptor-linked tyrosine phosphatases as the human leukocyte common antigen. CD45.