RESUMEN
PRM-151, recombinant human Pentraxin-2 (PTX-2) also referred to as serum amyloid P (SAP), is under development for treatment of fibrosis. A First-in-Human (FIH) trial was performed to assess the safety, tolerability, and pharmacokinetics of single ascending intravenous doses of PRM-151 administered to healthy subjects, using a randomized, blinded, placebo controlled study design. Each cohort included three healthy subjects (PRM-151:placebo; 2:1). SAP levels were assessed using a validated ELISA method, non-discriminating between endogenous and exogenous SAP. At a dose level of 10 mg/kg, at which a physiologic plasma level of SAP was reached, two additional healthy volunteers and three pulmonary fibrosis (PF) patients were enrolled enabling comparison of the pharmacokinetic SAP profile between healthy volunteers and PF patients. In addition, the percentage of fibrocytes (CD45+/Procollagen-1+ cells) in whole blood samples was assessed to demonstrate biological activity of PRM-151 in the target population. PRM-151 administration was generally well tolerated. In two pulmonary fibrosis patients non-specific, transient skin reactions (urticaria and erythema) were observed. PRM-151 administration resulted in a 6-to 13-fold increase in mean baseline plasma SAP levels at dose levels of 5, 10, and 20 mg/kg. The estimated t1/2 of PRM-151 in healthy volunteers was 30 h. Pharmacokinetic profiles were comparable between healthy volunteers and PF patients. PRM-151 administration resulted in a 30-50% decrease in fibrocyte numbers 24 h post-dose. This suggests that administration of PRM-151 may be associated with a reduction of fibrocytes in PF patients, a population for which current pharmacotherapeutic options are limited. The pharmacological action of PRM-151 should be confirmed in future research.
Asunto(s)
Proteínas de Homeodominio/administración & dosificación , Fibrosis Pulmonar/tratamiento farmacológico , Componente Amiloide P Sérico/administración & dosificación , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Semivida , Proteínas de Homeodominio/efectos adversos , Proteínas de Homeodominio/farmacocinética , Humanos , Masculino , Persona de Mediana Edad , Fibrosis Pulmonar/fisiopatología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética , Componente Amiloide P Sérico/efectos adversos , Componente Amiloide P Sérico/farmacocinética , Adulto JovenRESUMEN
The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.
Asunto(s)
Antígeno-1 Asociado a Función de Linfocito/metabolismo , Sitio Alostérico/genética , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Adhesión Celular/genética , Células Clonales , Cricetinae , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Cinética , Ligandos , Antígeno-1 Asociado a Función de Linfocito/fisiología , Linfocitos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Resonancia por Plasmón de SuperficieRESUMEN
The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.
Asunto(s)
Antígenos CD18/química , Molécula 1 de Adhesión Intercelular/química , Antígeno-1 Asociado a Función de Linfocito/química , Sitio Alostérico , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Adhesión Celular , Cristalografía por Rayos X , Humanos , Ligandos , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/fisiología , Antígeno de Macrófago-1/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , TransfecciónRESUMEN
The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.
Asunto(s)
Regulación hacia Abajo/inmunología , Proteínas Nucleares , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Linfocitos T/enzimología , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Anticuerpos Monoclonales/farmacología , Complejo CD3/inmunología , Células COS , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/inmunología , Genes Reporteros , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFATC , Fosforilación , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirosina/fisiología , Proteína Tirosina Quinasa ZAP-70RESUMEN
Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation. We show that Cbl overexpression in 293T embryonic kidney and Jurkat T-lymphocyte cells led to a dramatic reduction in the active pool of Fyn; this was seen as a reduction in Fyn autophosphorylation, reduced phosphorylation of in vivo substrates, and inhibition of transcription from a Src-family kinase response element linked to a luciferase reporter. Importantly, a Fyn mutant (FynY528F) relieved of intramolecular repression was still negatively regulated by Cbl. The Cbl-dependent negative regulation of Fyn did not appear to be mediated by inhibition of Fyn kinase activity but was correlated with enhanced protein turnover. Consistent with such a mechanism, elevated levels of Fyn protein were observed in cell lines derived from Cbl(-/-) mice compared to those in wild-type controls. The effects of Cbl on Fyn were not observed when the 70ZCbl mutant protein was analyzed. Taken together, these observations implicate Cbl as a component in the negative regulation of Fyn and potentially other Src-family kinases, especially following kinase activation. These results also suggest that protein degradation may be a general mechanism for Cbl-mediated negative regulation of activated tyrosine kinases.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Sustitución de Aminoácidos , Animales , Línea Celular , Humanos , Riñón , Cinética , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-fyn , Proto-Oncogenes , Proteínas Recombinantes/metabolismo , Linfocitos T , TransfecciónRESUMEN
The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Transporte Biológico , Secuencia Conservada , Regulación hacia Abajo , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Proteínas Fluorescentes Verdes , Hemaglutininas/genética , Hemaglutininas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/metabolismo , Ubiquitinas/metabolismoRESUMEN
The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.
Asunto(s)
Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Células COS , Secuencia Conservada , Proteínas de Unión al ADN , Evolución Molecular , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunoglobulina M/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Linfoma de Células B , Mutación , Proteínas Nucleares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Respuesta Sérica , Transducción de Señal , Quinasa Syk , Activación Transcripcional , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
The Cbl protooncoprotein has recently emerged as a component of tyrosine kinase-mediated signal transduction in a variety of cell types. Here, we discuss evidence that supports a role for Cbl as a novel negative regulator of immune receptor signaling, and present models for its mode of function.
Asunto(s)
Proteínas Proto-Oncogénicas/inmunología , Receptores Inmunológicos/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Transducción de Señal/inmunologíaRESUMEN
The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.
Asunto(s)
Células Dendríticas/inmunología , Glicoproteínas de Membrana , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T/inmunología , Células 3T3 , Animales , Antígenos T-Independientes/inmunología , Dermatitis por Contacto/inmunología , Haptenos/inmunología , Hemocianinas/inmunología , Hipersensibilidad Tardía/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ligando OX40 , Ovalbúmina/inmunología , Receptores del Factor de Necrosis Tumoral/genética , Factores de Necrosis TumoralRESUMEN
The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867-8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk. Co-expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-zeta. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-zeta phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.
Asunto(s)
Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Sitios de Unión , Unión Competitiva , Regulación Enzimológica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Fosfopéptidos/metabolismo , Fosfotirosina/metabolismo , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes de Fusión/metabolismo , Quinasa Syk , Dominios Homologos srcRESUMEN
The Cbl protooncogene product has emerged as a negative regulator of receptor and nonreceptor tyrosine kinases. We recently demonstrated that oncogenic Cbl mutants upregulate the endogenous tyrosine kinase signaling machinery when expressed in the NIH 3T3 cells, and identified the platelet-derived growth factor receptor-alpha (PDGFRalpha) as one of the tyrosine kinases targeted by these oncogenes. These findings suggested a role for the normal Cbl protein in negative regulation of the PDGFRalpha. However, the mechanism of such negative regulation remained to be determined. Here we show that overexpression of the wild-type Cbl enhances the ligand-induced ubiquitination of the PDGFRalpha. Concomitantly, the PDGFRalpha in Cbl-overexpressing cells undergoes a faster ligand-induced degradation compared with that in the control cells. These results identify a role for Cbl in the regulation of ligand-induced ubiquitination and degradation of receptor tyrosine kinases and suggest one potential mechanism for evolutionarily conserved negative regulatory influence of Cbl on tyrosine kinases.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Transducción de Señal , Células 3T3 , Animales , Ratones , Proteína Oncogénica v-cbl , Ubiquitinas/metabolismoRESUMEN
Cbl has emerged as a novel signal transducing protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Identified as the protein product of the c-cbl proto-oncogene, the cellular homolog to the transforming gene of a murine retrovirus, Cbl comprises an N-terminal transforming region (Cbl-N), which contains a phosphotyrosine binding (PTB) domain, and a C-terminal modular region (Cbl-C) containing a RING finger motif, a large proline-rich region and a leucine zipper. Deletion of Cbl-C or small deletions N-terminal to the RING finger render Cbl oncogenic, whereas wild type Cbl is non-transforming, even if overexpressed. Cbl serves as a substrate of both receptor and non-receptor tyrosine kinases, and binds to adaptor proteins Grb2, Crk and the p85 subunit of PI-3-kinase. Additionally, both Caenorhabditis elegans and Drosophila Cbl homologs, SLI-1 and D-Cbl, respectively, have been identified as negative regulators of the LET-23/DER receptor tyrosine kinases. Finally, oncogenic mutants of Cbl, when expressed in fibroblasts, upregulate the signaling cascade downstream of the platelet-derived growth factor receptor alpha in a Cbl-PTB domain-dependent manner. Together, these findings position Cbl as a central player in the regulation of tyrosine kinase signaling pathways. Identification of the Cbl-PTB domain binding motifs on tyrosine kinases and elucidation of the mechanisms of Cbl's negative regulatory effect may provide a new avenue to control tyrosine kinases for therapeutic purposes.
Asunto(s)
Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Ubiquitina-Proteína Ligasas , Animales , Caenorhabditis elegans , Drosophila , Humanos , Proteínas Tirosina Quinasas/fisiología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cblRESUMEN
Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.
Asunto(s)
Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ubiquitina-Proteína Ligasas , Regulación hacia Arriba/fisiología , Células 3T3 , Animales , División Celular , Transformación Celular Neoplásica , Humanos , Ratones , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Transducción de Señal/fisiología , Dominios Homologos srcRESUMEN
The c-cbl protooncogene was first identified as the cellular homologue of a viral oncogene v-cbl that induces pre-B lymphomas and myeloid leukemias in mice. Until recently, the biochemical basis for Cbl's transforming potential and its physiological role remained unclear. However, a convergence of biochemical studies in mammalian cells and genetic studies in C. elegans and Drosophila has now identified Cbl as a negative regulator of tyrosine kinase signaling. The N-terminal transforming region of Cbl (Cbl-N) and an adjacent RING finger domain are the elements most conserved during evolution. The Cbl-N region has now been shown to contain a novel phosphotyrosine-binding (PTB) domain that directly interacts with autophosphorylated tyrosine kinases via a D(N/D)XpY motif. A critical role of the PTB domain in Cbl function is demonstrated by the localization of a loss-of-function mutation in C. elegans Cbl homologue SLI-1 within this region. The corresponding mutation in human Cbl inactivates the PTB domain function and abrogates Cbl-mediated regulation of tyrosine kinase function. Recent studies have also identified a novel signaling pathway initiated by the interaction of mammalian Cbl proteins with the SH2 domains of Crk adaptor molecules, which results in Cbl's linkage with C3G, a guanine nucleotide exchange protein for Rap1 family of small G-proteins. Presently, Rap1 is thought to antagonize Ras function, although Rap1-specific targets have emerged recently. Thus, recent advances have firmly placed the little known protooncoprotein Cbl on the center stage of tyrosine kinase-mediated signal transduction.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Transformación Celular Neoplásica/genética , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cblRESUMEN
The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)292) specifically inhibited binding of ZAP-70 to Cbl-N. A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.
Asunto(s)
Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Células Jurkat , Ratones , Datos de Secuencia Molecular , Proteína Oncogénica v-cbl , Fosforilación , Proteína Tirosina Quinasa ZAP-70 , Dominios Homologos srcRESUMEN
The protooncogene product Cbl has emerged as a novel signal transduction protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Recently, we and others have reported the activation-dependent association of Cbl with the Syk and ZAP-70 tyrosine kinases through presently undefined mechanisms. Potential Src homology 2 and 3 domain binding sites within the C-terminal half of Cbl mediate in vivo interactions with several signaling proteins; however, the N-terminal transforming region (Cbl-N) lacks recognizable catalytic or protein interaction motifs. Here, we show that in vitro Cbl-N (amino acids 1-357) but not Cbl-C (amino acids 358-906) binds to ZAP-70 in a T cell-activation-dependent manner. A point mutation in Cbl-N, G306E, corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue, SLI-1, severely compromised Cbl-N/ZAP-70 binding. Cbl-N/ZAP-70 binding was direct and phosphotyrosine-dependent, thus identifying a phosphotyrosine-binding domain within the transforming region of Cbl. In vivo, Cbl-N expressed in T cells selectively associated with the ZAP-70/zeta complex. These results identify a novel mechanism for the direct participation of the N-terminal region of Cbl in ZAP-70 signal transduction, and suggest a biochemical mechanism for the leukemogenicity of the oncogene v-cbl through potential interaction with proliferation-related phosphotyrosyl proteins.