RESUMEN

BACKGROUND: Fibroblast growth factors (FGFs) play important roles in multiple cancers by supporting tumor growth and angiogenesis. FP-1039 (GSK3052230) is a FGF ligand trap consisting of the extracellular domain of FGF receptor 1 (FGFR1) fused with the Fc region of IgG1. FP-1039 binds and neutralizes multiple FGFs that normally bind FGFR1. The primary objective of this phase I study was to evaluate the safety and tolerability of FP-1039. PATIENTS AND METHODS: Eligible patients with metastatic or locally advanced solid tumors for which standard treatments were ineffective were treated with weekly doses of FP-1039 for 4 weeks, followed by 2 weeks observation. RESULTS: Thirty-nine subjects received a mean of 6 infusions of FP-1039 at doses ranging from 0.5 to 16 mg/kg weekly, with no maximally tolerated dose identified. Grade 3 or greater treatment emergent adverse events were uncommon. Four dose-limiting toxicities were reported at doses of 0.75 mg/kg (urticaria), 1 mg/kg (intestinal perforation and neutropenia), and 16 mg/kg (muscular weakness). Drug exposure was dose proportional, and the terminal elimination half-life was 2.6-3.9 days following a single dose. Target engagement as measured by low free plasma FGF2 levels was achieved. FGF pathway dysregulation was uncommon. No objective responses were observed. CONCLUSION: In nonselected cancer patients with advanced disease, treatment with FP-1039 was well tolerated and toxicities associated with small molecule drugs that inhibit FGFR tyrosine kinases, including hyperphosphatemia, were not observed. Further studies of FP-1039 in patients selected for FGF pathway dysregulation, who are most likely to benefit, are now underway.

Asunto(s)

Antineoplásicos/uso terapéutico , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Inmunoglobulina G/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas de Fusión Oncogénica/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/uso terapéutico , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunoglobulina G/efectos adversos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/efectos adversos , Proteínas de Fusión Oncogénica/farmacocinética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión

RESUMEN

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

Asunto(s)

Diseño de Fármacos , Proteínas Proto-Oncogénicas c-jun/genética , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Proteínas Recombinantes de Fusión/genética , Células 3T3 , Animales , Línea Celular , Dimerización , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Ratones , Unión Proteica , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.

Asunto(s)

Colágeno/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Dominios Homologos src , Proteínas de Fusión bcr-abl/genética , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Fosfotirosina/metabolismo , Células Tumorales Cultivadas

RESUMEN

We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

Asunto(s)

Células CHO/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Genes fos , Técnicas Genéticas , Insulina/farmacología , Proteínas Quinasas Activadas por Mitógenos , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Oligonucleótidos/química , Fosforilación , ARN Mensajero/efectos de los fármacos , Juego de Reactivos para Diagnóstico , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Análisis de Secuencia de ADN , Factores de Tiempo

RESUMEN

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Asunto(s)

Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Monoéster Fosfórico Hidrolasas/metabolismo , Células 3T3 , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Transportador de Glucosa de Tipo 4 , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Dominios Homologos src

RESUMEN

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.

Asunto(s)

Quinasas CDC2-CDC28 , Ciclo Celular , Transformación Celular Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Apoptosis , División Celular , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , ADN/biosíntesis , Activación Enzimática , Oncogenes , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Albúmina Sérica Bovina , Transducción de Señal , Factores de Tiempo , Transformación Genética

RESUMEN

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors. Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms. Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction. We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains. From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.

Asunto(s)

Histidina/análogos & derivados , Isoenzimas/metabolismo , Péptidos/metabolismo , Fosfotirosina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Cromatografía Líquida de Alta Presión , Polarización de Fluorescencia , Histidina/metabolismo , Isoenzimas/química , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Fosfolipasa C gamma , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/química

RESUMEN

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

Asunto(s)

Insulina/farmacología , Oocitos/citología , Monoéster Fosfórico Hidrolasas/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Dominios Homologos src , Animales , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Xenopus

RESUMEN

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.

Asunto(s)

Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Animales , Proteínas Sanguíneas/genética , Células COS , Activación Enzimática , Inositol Polifosfato 5-Fosfatasas , Membranas Artificiales , Fosfatidilinositol 3-Quinasas , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Mutación Puntual , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt

RESUMEN

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.

Asunto(s)

Proteínas Quinasas Activadas por Mitógenos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Compartimento Celular , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Chlorocebus aethiops , Cartilla de ADN/química , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Prenilación de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Relación Estructura-Actividad , Proteína de Unión al GTP cdc42

RESUMEN

BACKGROUND: Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS: The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS: These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B , Secuencia de Bases , Caenorhabditis elegans , Línea Celular Transformada , Chlorocebus aethiops , Clonación Molecular , Receptores ErbB/genética , Proteína Adaptadora GRB2 , Humanos , Inositol Polifosfato 5-Fosfatasas , Activación de Linfocitos , Datos de Secuencia Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Proteínas/genética , Conejos , Transducción de Señal

RESUMEN

The phosphotyrosine binding (PTB) domain specifically binds to tyrosine-phosphorylated proteins, but differs in structure and mechanism of action from the SH2 domain family. We quantitated the affinity, specificity, and kinetics of the interaction of the SHC PTB domain with a sequence motif, asparagine-X-X-phosphotyrosine (NXX(pY)), found in several receptor tyrosine kinases and oncogenic proteins. PTB domain-mediated interaction with the NXX(pY) motif of c-ErbB2 was characterized by similar overall affinity but slower kinetics than that reported for SH2 domains. This suggested that unlike SH2 domains, PTB domains may not rapidly exchange among associated proteins. Furthermore, when directly and quantitatively compared, PTB domain binding specificity did not significantly overlap with a panel of seven SH2 domains. Thus, signaling pathways involving PTB and SH2 domain-mediated interactions can be regulated separately. Finally, our data define the minimal SHC PTB domain binding motif as NXX(pY), not NPX(pY) as suggested by other authors, and suggest a high affinity motif, hydrophobic residue-(D/E)-N-X-X-pY-(W/F), found in the Trk and ErbB receptor tyrosine kinase families. We conclude that PTB domains mediate specific protein-protein interactions independent from those mediated by SH2 domains.

Asunto(s)

Asparagina/metabolismo , Fosfotirosina/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cinética , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento/química , Transducción de Señal

RESUMEN

Src homology 2 (SH2) domains mediate assembly of signaling complexes by binding specifically to tyrosine-phosphorylated proteins. A phosphotyrosine binding (PTB) domain has been identified which also binds specifically to tyrosine-phosphorylated targets, but is structurally different from SH2 domains. Expression cloning was used to identify targets of PTB domains. PTB domains bound to phosphotyrosine within a sequence motif, asparagine-X1-X2-phosphotyrosine (where X represents any amino acid), that is found in many signaling proteins and is not recognized by SH2 domains. Mutational studies indicated that high affinity binding of PTB domains may require a specific conformation of the motif.

Asunto(s)

Fosfopéptidos/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Unión Competitiva , Línea Celular , Humanos , Datos de Secuencia Molecular , Fosfotirosina , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Tirosina/metabolismo

RESUMEN

Src homology 2 (SH2) domains bind specifically to tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes. A protein domain was identified that bound specifically to the tyrosine-phosphorylated form of its target protein but differs from known SH2 sequences. Phosphotyrosine-binding (PTB) domains were found in two proteins: SHC, a protein implicated in signaling through Ras; and SCK, encoded by a previously uncharacterized gene. The PTB domain of SHC specifically bound to a tyrosine-phosphorylated 145-kilodalton protein. PTB domains are an alternative to SH2 domains for specifically recruiting tyrosine-phosphorylated proteins into signaling complexes and are likely to take part in signaling by many growth factors.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Tirosina/análogos & derivados , Células 3T3 , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina , Factor de Crecimiento Derivado de Plaquetas/farmacología , Unión Proteica , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas/química , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismo

RESUMEN

The mechanisms by which growth factors and oncogenic agents activate phosphatidylinositol 3-kinase (PI3 kinase) are unknown. Previously, we reported that the 85-kDa regulatory subunit of PI3 kinase is tyrosine-phosphorylated both in vitro by the platelet-derived growth factor beta-receptor (PDGFR) tyrosine kinase and in fibroblasts in response to PDGF. As a first step in determining the role of tyrosine phosphorylation in PDGF signaling through PI3 kinase, we investigated which tyrosines on p85 are phosphorylated by the PDGFR. Recombinant p85 was phosphorylated with recombinant PDGF receptors, and tryptic phosphopeptides were purified by HPLC and analyzed by Edman degradation. By this approach and by mutational analysis, Y508 was identified as the major in vitro phosphorylation site. Tryptic phosphopeptide mapping demonstrated Y508 to also be phosphorylated in vivo in COS cells. Comparison of these data with a previous report [Hayashi, H., Nishioka, Y., Kamohara, S., Kanai, F., Ishii, K., Fukui, Y., Shibasaki, F., Takenawa, T., Kido, H., Katsunuma, N., & Ebina, Y. (1993) J. Biol. Chem. 268, 7107-7117] suggests that p85 is phosphorylated differently by the PDGF and insulin receptor tyrosine kinases. Therefore, p85 may be regulated differently by PDGF and insulin. Mapping of phosphorylation sites on p85 may lead to new insights into the regulation of signal transduction through PI3 kinase.

Asunto(s)

Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Ratones , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Proteínas Recombinantes/metabolismo

RESUMEN

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.

Asunto(s)

Fosfotransferasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Superficie Celular/metabolismo , Células 3T3 , Adenilil Imidodifosfato/farmacología , Animales , Línea Celular , Células Clonales , Ácido Edético/farmacología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Insectos , Cinética , Sustancias Macromoleculares , Ratones , Modelos Biológicos , Peso Molecular , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/aislamiento & purificación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Unión Proteica , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/aislamiento & purificación , Receptores del Factor de Crecimiento Derivado de Plaquetas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor. Experiments with receptor mutants and with short peptides derived from the kinase insert region of the PDGF receptor showed that the recombinant p85 binds to a well-defined phosphotyrosine-containing sequence of the receptor. p85 appears to be the subunit of PI3-kinase that links the enzyme to the ligand-activated receptor.

Asunto(s)

Fosfotransferasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Superficie Celular/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Línea Celular , Clonación Molecular , ADN , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico

RESUMEN

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.

Asunto(s)

Fosfotransferasas/metabolismo , Receptores de Superficie Celular/metabolismo , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Sitios de Unión , Línea Celular , Insectos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peso Molecular , Fosfatidilinositol 3-Quinasas , Fosfopéptidos/síntesis química , Fosfopéptidos/aislamiento & purificación , Fosfotransferasas/aislamiento & purificación , Fosfotirosina , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Receptores del Factor de Crecimiento Derivado de Plaquetas

RESUMEN

Some human tumor cell lines express the c-sis gene, the proto-oncogene of the transforming gene v-sis, and produce platelet-derived growth factor, which may contribute to carcinogenesis by autocrine or paracrine mechanisms. Here we demonstrate that c-sis expression in some human glioma and osteosarcoma cell lines can be blocked by agents that increase cellular cyclic adenosine monophosphate (cAMP). Forskolin, 8-bromocyclic AMP, cholera toxin, and prostaglandin E1 reduced c-sis mRNA in these cells by up to 90%. c-sis transcription rates were reduced by agents that increase cAMP; the stability of c-sis mRNA was unaffected. The possible therapeutic value of blocking the expression of tumor growth factor genes pharmacologically warrants further study.

Asunto(s)

AMP Cíclico/fisiología , Regulación de la Expresión Génica , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Colforsina/farmacología , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-sis , Receptores de Superficie Celular/análisis , Receptores del Factor de Crecimiento Derivado de Plaquetas , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.

Asunto(s)

ADN/biosíntesis , Músculo Liso Vascular/metabolismo , Toxina del Pertussis , Serotonina/farmacología , Factores de Virulencia de Bordetella/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Calcio/análisis , Calcio/metabolismo , Bovinos , Células Cultivadas/efectos de los fármacos , AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , ADN/efectos de los fármacos , Hidrólisis , Mitosis/efectos de los fármacos , Músculo Liso Vascular/citología , Fosfatidilinositoles/metabolismo