RESUMEN
The leukemic oncogene Tel/PDGFRbeta, was inducibly expressed in embryonic stem (ES) cells and the phenotypic and molecular changes occurring during hematopoietic differentiation investigated. Expression of Tel/PDGFRbeta resulted in an inability of ES cells to self-renew and caused a significant increase in myelopoiesis with a corresponding decrease in erythropoiesis. Analysis of gene expression patterns indicated a dramatic alteration in the levels of genes associated with self-renewal and differentiation, especially myelomonocytic genes in Tel/PDGFRbeta-expressing cells. This study indicates Tel/PDGFRbeta drives myelopoiesis by altering expression of genes involved in hematopoiesis and demonstrates the potential of this stem cell system to study oncogene-induced pathogenesis.
Asunto(s)
Células Madre Embrionarias/metabolismo , Mielopoyesis , Proteínas de Fusión Oncogénica/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Hematopoyesis/genética , Factor Inhibidor de Leucemia/farmacología , Ratones , Células Mieloides/citologíaRESUMEN
ES (embryonic stem) cell lines are derived from the epiblast of pre-implantation embryos and like the inner cell mass cells from which they are derived exhibit the remarkable property of pluripotency, namely the ability to differentiate into all cell lineages comprising the adult organism. ES cells and their differentiated progeny offer tremendous potential to regenerative medicine, particularly as cellular therapies for the treatment of a wide variety of chronic disorders, such as Type 1 diabetes, Parkinson's disease and retinal degeneration. In order for this potential to be realized, a detailed understanding of the molecular mechanisms regulating the fundamental properties of ES cells, i.e. pluripotency, proliferation and differentiation, is required. In the present paper, we review the evidence that PI3K (phosphoinositide 3-kinase)-dependent signalling plays a role in regulation of both ES cell pluripotency and proliferation.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Diferenciación Celular , División Celular , Homeostasis , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Transducción de SeñalRESUMEN
The purpose of this study was to rapidly determine, without the use of extensive animal studies, whether biological activity is retained after spray drying insulin with two excipients, lactose and xanthan gum. This was achieved by the detection of protein kinase B (PKB), which is activated by phosphorylation in response to insulin binding to cellular receptors. A myeloid cell line was cultured and stimulated with the reconstituted insulin powders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then utilised to allow in-vitro detection of phosphorylated PKB using an anti-phospho-PKB antibody. A single band specific to phosphorylated PKB was found on the Western blots, indicating that the active conformation of insulin was retained when spray dried in combination with lactose and with xanthan gum over the spray-drying inlet temperature range of 110-170 degrees C. Evidence of inactivation/denaturation was observed when insulin was spray dried at an inlet temperature of 200 degrees C. The assay may be of use as a more rapid and economic means to screen insulin formulations for inhalation and other purposes as opposed to conventional monitoring of blood glucose levels in animals.
Asunto(s)
Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacología , Insulina/administración & dosificación , Insulina/farmacología , Desecación , Electroforesis en Gel de Poliacrilamida , Excipientes , Lactosa , Microscopía Electrónica de Rastreo , Fosforilación , Polisacáridos Bacterianos , TemperaturaRESUMEN
We have demonstrated previously that class I(A) phosphoinositide 3-kinases play a major role in regulation of interleukin-3 (IL)-3-dependent proliferation. Investigations into the downstream targets involved have identified the MAPK cascade as a target. Expression of Deltap85 and incubation with LY294002 both inhibited IL-3-induced activation of Mek, Erk1, and Erk2. This was most pronounced during the initial phase of Erk activation. The Mek inhibitor, PD98059, blocked IL-3-driven proliferation, an effect enhanced by Deltap85 expression, suggesting that inhibition of Mek and Erks by Deltap85 contributes to the decrease in IL-3-induced proliferation in these cells but that additional pathways may also be involved. To investigate the mechanism leading to decreased activation of Erks, we investigated effects on SHP2 and Gab2, both implicated in IL-3 regulation of Erk activation. Expression of Deltap85 led to a reduction in SHP2 tyrosine phosphorylation and its ability to interact with Grb2 and Gab2 but increased overall tyrosine phosphorylation of Gab2. LY294002 did not perturb SHP2 interactions, potentially related to differences in the effects of these inhibitors on levels of phosphoinositides. These results imply that the regulation of Erks by class I(A) phosphoinositide 3-kinase may contribute to IL-3-driven proliferation and that both SHP2 and Gab2 are possibly involved in this regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Interleucina-3/farmacología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosfoproteínas/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Animales , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/metabolismo , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Proteína Adaptadora GRB2 , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Eliminación de SecuenciaRESUMEN
p46(Shc) and p52(Shc) become heavily tyrosine phosphorylated in response to interleukin 3 (IL-3) treatment. We have investigated the potential of Shc to integrate IL-3 signalling pathways and demonstrate that Shc associates with the beta subunits of the human (betac) and murine (Aic2A) IL-3 receptors, SHIP and Gab2 following IL-3 stimulation. The interaction between Shc and the IL-3 receptor beta chains was direct, mediated by both the SH2 and PTB domains. Interaction with SHIP was via the Shc PTB domain and the Shc SH2 domain mediated the interaction with Gab2. Phosphopeptide competition studies suggest that the SH2 domain interacts primarily with tyrosine 612 of betac (610 of Aic2A), and the PTB domain with tyrosine 577 of betac (575 of Aic2A). PTB binding to IL-3R beta chains was of highest affinity, and appeared to play the primary role in binding. These findings suggest that Shc may play an important role in coordinately integrating IL-3 signalling pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Interleucina-3/farmacología , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal/efectos de los fármacos , Dominios Homologos src , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Humanos , Leucemia Eritroblástica Aguda/patología , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Tirosina/metabolismoRESUMEN
Activation of phosphoinositide-3 kinases (PI3Ks), their downstream target protein kinase B (PKB), and phosphorylation of Bad have all been implicated in survival signaling in many systems. However, it is not known whether these events are sufficient or necessary to universally prevent apoptosis. To address this issue, we have used three different factor-dependent hemopoietic cell lines, MC/9, BaF/3, and factor-dependent (FD)-6, which respond to a range of cytokines, to investigate the relationship between PI3K, PKB, and Bad activity with survival. The cytokines IL-3, IL-4, stem cell factor (SCF), GM-CSF, and insulin all induced the rapid and transient activation of PKB in responsive cell lines. In all cases, cytokine-induced PKB activation was sensitive to inhibition by the PI3K inhibitor, LY294002. However, dual phosphorylation of the proapoptotic protein Bad was found not to correlate with PKB activation. In addition, we observed cell-type-specific differences in the ability of the same cytokine to induce Bad phosphorylation. Whereas IL-4 induced low levels of dual phosphorylation of Bad in FD-6, it was unable to in MC/9 or BaF/3. Insulin, which was the most potent inducer of PKB in FD-6, induced barely detectable Bad phosphorylation. In addition, the ability of a particular cytokine to induce PKB activity did not correlate with its ability to promote cell survival and/or proliferation. These data demonstrate that, in hemopoietic cells, activation of PKB does not automatically confer a survival signal or result in phosphorylation of Bad, implying that other survival pathways must be involved.
Asunto(s)
Proteínas Portadoras/metabolismo , Citocinas/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , División Celular/inmunología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Cromonas/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Ratones , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Serina/metabolismo , Proteína Letal Asociada a bclRESUMEN
Interleukin-3 (IL-3) acts as both a growth and survival factor for many hemopoietic cells. IL-3 treatment of responsive cells leads to the rapid and transient activation of Class IA phosphoinositide-3-kinases (PI3Ks) and the serine/threonine kinase Akt/protein kinase B (PKB) and phosphorylation of BAD. Each of these molecules has been implicated in anti-apoptotic signaling in a wide range of cells. Using regulated expression of dominant-negative p85 (Deltap85) in stably transfected IL-3-dependent BaF/3 cells, we have specifically investigated the role of class IA PI3K in IL-3 signaling. The major functional consequence of Deltap85 expression in these cells is a highly reproducible, dramatic reduction in IL-3-induced proliferation. Expression of Deltap85 reduces IL-3-induced PKB phosphorylation and activation and phosphorylation of BAD dramatically, to levels seen in unstimulated cells. Despite these reductions, the levels of apoptosis observed in the same cells are very low and do not account for the reduction in IL-3-dependent proliferation we observe. These results show that Deltap85 inhibits both PKB activity and BAD phosphorylation without significantly affecting levels of apoptosis, suggesting that there are targets other than PKB and BAD that can transmit survival signals in these cells. Our data indicate that the prime target for PI3K action in IL-3 signaling is at the level of regulation of proliferation.
Asunto(s)
Apoptosis , Proteínas Portadoras/metabolismo , Interleucina-3/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Bovinos , División Celular , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Proteína Letal Asociada a bclRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Secuencia de Aminoácidos , Androstadienos/farmacología , Sitios de Unión , Antígenos CD28/metabolismo , Cromonas/farmacología , Regulación hacia Abajo , Humanos , Células Jurkat , Datos de Secuencia Molecular , Morfolinas/farmacología , Mutación , Mapeo Peptídico , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosfopéptidos/análisis , Fosforilación , Fosfoserina/metabolismo , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Transducción de Señal , WortmaninaRESUMEN
DSM-IV states that Asperger Disorder may be distinguished from Autistic Disorder by a lack of a delay in early language development. The aim of this study was to establish whether the presence or absence of early language delay would predict autistic symptomatology in children diagnosed with a PDD/autism spectrum disorder. Forty-six language-delayed and 62 normal language onset individuals (M age 11 years) were compared on ICD-10 research criteria and DSM-IV criteria, receptive language, and developmental history variables. Retrospective data were also obtained to determine whether language onset predicted autism symptomatology when young (< 6 years). We found that early language delay predicts more autistic symptomatology when young, but not at an older age. Early language delay is also associated with developmental motor milestone delays and lower receptive language abilities. The results question the use of early language delay as a valid discriminating variable between PDD subgroups.
Asunto(s)
Trastorno Autístico/diagnóstico , Trastornos Generalizados del Desarrollo Infantil/diagnóstico , Trastornos del Desarrollo del Lenguaje/diagnóstico , Adolescente , Trastorno Autístico/psicología , Niño , Trastornos Generalizados del Desarrollo Infantil/psicología , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Trastornos del Desarrollo del Lenguaje/psicología , Masculino , Pronóstico , Escalas de Valoración Psiquiátrica , Trastornos Psicomotores/diagnóstico , Trastornos Psicomotores/psicología , SíndromeRESUMEN
We have observed previously the co-immunoprecipitation of the p85 subunit of phosphatidylinositol-3 kinase (PI3K) and SHP2 in murine lymphohemopoietic cells after stimulation with interleukin-3. We have investigated this interaction in more detail and now report the identification of a potentially novel 100-kDa protein (termed p100), which is inducibly phosphorylated on tyrosine after interleukin-3 treatment and which co-immunoprecipitates with both p85 PI3K and SHP2. The Src homology region 2 domains of both p85 and SHP2 appear to mediate their interactions with p100. Sequential precipitation analyses suggest that these interactions are direct and do not involve Grb2, and that the same p100 protein, or a portion of it, interacts with both p85 and SHP2, implying that p100 may serve to link these two proteins. Far Western blotting with both full-length p85 and isolated p85 Src homology region 2 domains supports this view. Interestingly, p100 also appears to be a substrate for the SHP2 phosphatase activity. In addition, p100 is precipitated by Grb2-glutathione S-transferase fusion proteins, an interaction largely mediated by the Grb2 SH3 domains. p100 appears to be distinct from JAK2, Vav, STAT5, and c-Cbl. Although largely cytosolic, p100 can be detected associated with SHP2 and PI3K in crude membrane fractions after interleukin-3 stimulation. We propose that p100 plays a role as an adaptor molecule, linking PI3K and SHP2 in IL-3 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Células de la Médula Ósea/enzimología , Interleucina-3/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Células Cultivadas , Proteína Adaptadora GRB2 , Péptidos y Proteínas de Señalización Intracelular , Ratones , Fosforilación , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas/metabolismo , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
The cytoplasmic tyrosine phosphatases, SHP1 and SHP2, are implicated in the control of cellular proliferation and survival. Here we demonstrate that both SHP1 and SHP2 associate with the betac subunit of the human interleukin-3 (IL-3) receptor following IL-3 stimulation and that the src homology region 2 (SH2) domains of these phosphatases mediate this interaction. Sequential immunoprecipitation analyses suggest this interaction is direct. Competition studies, using phosphotyrosine-containing peptides based on sequences surrounding key tyrosine residues within betac, suggest that phosphorylation of tyrosine 612 is the key event mediating the association of betac with SHP1 and SHP2. However, inhibition of SHP2 binding to betac, did not prevent tyrosine phosphorylation of SHP2. Interestingly, this same phosphopeptide served as a substrate for the tyrosine phosphatase activity of both SHP1 and SHP2. Binding of these protein-tyrosine phosphatases to the IL-3 receptor may regulate IL-3 signal transduction pathways, both through their catalytic activity and through the recruitment of other molecules to the receptor complex.
Asunto(s)
Interleucina-3/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal , Sitios de Unión , Línea Celular , Humanos , Interleucina-3/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.
Asunto(s)
Monocitos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Cromonas/farmacología , Clonación Molecular , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/clasificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Wortmanina , Proteínas ras/metabolismo , Dominios Homologos srcRESUMEN
Insulin receptor substrate 1 (IRS-1), and its structural relative IRS-2, are both phosphorylated on tyrosine following treatment of cells with interleukin-4 (IL-4) and insulin. We have investigated whether both IRS-1 and IRS-2 are expressed in murine lymphohemopoietic cells. T and B lymphocytes and macrophages from primary cultures expressed only IRS-2, which became phosphorylated on tyrosine following stimulation with both IL-4 and insulin. Likewise, the murine myeloid cell line FD-5 expressed only IRS-2, which was tyrosine phosphorylated in response to IL-4 and insulin, as well as interleukin-3 and granulocyte-macrophage colony stimulating factor. Neither IRS-1 nor IRS-2 were expressed at detectable levels in primary bone marrow mast cells although these cells do respond to IL-4. Moreover, a factor-dependent lymphocyte cell line, CT.4S, which grows continuously in IL-4, did not express detectable levels of IRS-1 or IRS-2. IRS-2 from FD-5 cells stimulated with either IL-4 or insulin bound to glutathione S-transferase fusion proteins of the p85 subunit of phosphoinositol 3'-kinase, Grb2, and Syp, paralleling reported associations of IRS-1 with these molecules and indicating phosphorylation of the corresponding residues on IRS-2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Aviares , Citocinas/farmacología , Células Madre Hematopoyéticas/metabolismo , Linfocitos/metabolismo , Fosfoproteínas/metabolismo , Tirosina/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2 , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Interleucina-3/farmacología , Interleucina-4/farmacología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Peso Molecular , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismoRESUMEN
OBJECTIVE: To determine what clinical symptoms clinicians have been using to distinguish between Asperger's disorder (AsD) and autistic disorder (AD). METHOD: Parents of children and adolescents with high-functioning AD (n = 48) and AsD (n = 69) were given a structured interview based on DSM-III-R and ICD-10 diagnostic criteria. Information regarding early and current symptom presentation and family, developmental, and verbal mental age information were collected. Logistic regression analyses were conducted to determine which variables best predicted clinician's diagnosis. RESULTS: A number of clinical variables predicted diagnosis. Delayed language onset was the only variable of the family and developmental variables that predicted diagnosis. The AsD group was also significantly higher than the AD group in verbal mental age. CONCLUSION: Clinicians appear to be diagnosing AsD and AD on the basis of published research and case study accounts. The findings question whether DSM-IV and ICD-10 criteria adequately describe the AsD individual, particularly in the communication domain.
Asunto(s)
Trastorno Autístico/diagnóstico , Adolescente , Adulto , Trastorno Autístico/clasificación , Trastorno Autístico/genética , Trastorno Autístico/psicología , Niño , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Determinación de la Personalidad , Escalas de Valoración Psiquiátrica , Factores de Riesgo , SíndromeRESUMEN
Hematopoietic progenitor cells of the A/J strain of mice show a pronounced defect in the ability to form colonies or proliferate in response to interleukin-3 (IL-3). Comparison of immunoblots of A/J mast cells and of mast cells from the C57BL/6 strain that respond normally to IL-3 showed that, in both strains, a 125-kD band of the expected size was recognized by an antibody against the beta chain of the IL-3 receptor, the AIC2A molecule. However, in the C57BL/6 cells, there was an additional 110-kD species not seen in cells of the A/J strain. Analyses using bone marrow-derived mast cells from a panel of A/J x C57BL/6 and A/J x C57BL/6 recombinant inbred (RI) mice showed that the hypo-responsiveness to IL-3 is governed by a single gene. However, the absence of this 110-kD species in the A/J strain did not co-map with IL-3 hypo-responsiveness but did indeed map to the AIC2A genetic locus. These data show that this trait in the A/J strain was due to a polymorphism of the AIC2A gene unrelated to IL-3 hypo-responsiveness. Typing of the RI strains for the markers D14Mit98, D14Mitl4, and D14Mit133 mapped the locus determining hypo-responsiveness to IL-3 to the subtelomeric region of chromosome 14, the region that also bears the gene encoding the alpha chain of the IL-3 receptor (lL-3Ralpha). Immunofluorescence analyses indicated that IL-3Ralpha protein was undetectable on fresh bone marrow cells from A/J mice, although clearly detectable on cells from the responder C57BL/6 strain. However, IL-3Ralpha was readily detectable at normal levels on A/J mast cells generated by culture of A/J bone marrow cells in a combination of IL-3 and steel factor. Moreover, IL-3Ralpha on these A/J mast cells appears to be functional in that IL-3 stimulation of these cells results in tyrosine phosphorylation events characteristic of IL-3 signaling, including tyrosine phosphorylation of the beta chain of the IL-3 receptor, Jak-2 kinase, and SHPTP2. Collectively, these data indicate that the hypo-responsiveness of A/J mice to IL-3 is due to a defect in the gene encoding IL-3Ralpha and that, although this defect gives rise to reduced expression of alpha chain on primary bone marrow cells, this defect is not absolute and that, under certain circumstances, A/J cells can express functional receptors.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Interleucina-3/farmacología , Ratones Endogámicos A/genética , Receptores de Interleucina-3/deficiencia , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Polimorfismo Genético , Receptores de Interleucina-3/química , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-3/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Especificidad de la Especie , Factor de Células Madre/farmacologíaRESUMEN
Interleukin-13 (IL-13) and interleukin-4 (IL-4) are related in structure and function and are thought to share a common receptor component. We have investigated the signal transduction pathways activated by these two growth factors, as well as insulin, in cell-lines and primary cells of lymphohemopoietic origin. All three factors induced the tyrosine phosphorylation of a protein of 170 kDa (p170), which coimmunoprecipitated with the p85 subunit of P13'-kinase, via high affinity interactions mediated by the SH2 domains of p85. Antibodies raised against the entire insulin-receptor substrate-1 (IRS-1) protein immunoprecipitated p170 much less efficiently than they did IRS-1 from 3T3 cells. However, antibodies directed against the conserved pleckstrin homology domain of IRS-1 immunoprecipitated both p170 and IRS-1 with similar efficiency, suggesting they share structural similarities in this region. In lymphohemopoietic cells, IL-13, IL-4, and insulin failed to induce increased tyrosine phosphorylation of Shc, or its association with grb2, modification of Sos1, or activation of erk-1 and erk-2 mitogen-activated protein kinases, suggesting that p170 mediates downstream pathways distinct from those mediated by IRS-1. Both IL-13 and IL-4 induced low levels of tyrosine phosphorylation of Tyk-2 and Jak-1. IL-4 also activated the Jak-3-kinase, but, despite other similarities, IL-13 did not. Insulin failed to activate any of the known members of the Janus family of kinases. In that Jak-3 is reported to associate with the IL-2 gamma c chain, these data suggest that the IL-13 receptor does not utilize this subunit. However, both IL-13 and IL-4 induced tyrosine phosphorylation of the IL-4-140 kDa receptor chain, suggesting that this is a component of both receptors in these cells and accounts for the similarities in signaling pathways shared by IL-13 and IL-4.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-13/farmacología , Proteínas Quinasas Activadas por Mitógenos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células 3T3/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Proteína Adaptadora GRB2 , Células Madre Hematopoyéticas/fisiología , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/farmacología , Janus Quinasa 1 , Janus Quinasa 3 , Leucemia Eritroblástica Aguda/patología , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Plasmacitoma/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/fisiología , Receptores de Interleucina-13 , Receptores de Interleucina-4 , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales CultivadasRESUMEN
Binding of interleukin (IL)-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) to their high affinity cell surface receptors induces tyrosine phosphorylation of a similar set of protein substrates. We have identified one of these common substrates (p70) as the protein-tyrosine phosphatase SHPTP2. The Src homology 2 (SH2) domain of the adaptor protein Grb2 bound with high affinity to tyrosine-phosphorylated SHPTP2 following treatment of cells with IL-3 or GM-CSF, but not IL-4. This interaction was inhibited by two phosphotyrosine peptides, based on sequences within SHPTP2, which conform to the postulated consensus sequence for Grb2 SH2 recognition. Following treatment with IL-3 or GM-CSF, but not IL-4, SHPTP2 co-immunoprecipitated with antibodies directed against the p85 subunit of PI 3'-kinase. This was partially blocked by the same phosphopeptides that blocked Grb2-SH2 binding to SHPTP2. Importantly, treatment with IL-3 resulted in a 2-3-fold increase in SHPTP2 phosphatase activity. These results suggest that SHPTP2 may play an important role in integrating signals from the IL-3 and GM-CSF receptors to both Ras and PI 3'-kinase.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interleucina-3/farmacología , Interleucina-4/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Proteína Adaptadora GRB2 , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosfopéptidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotirosina , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Shc, grb2, and Son-of-sevenless (mSos1) proteins are potential upstream regulators of p21ras. We show that p52Shc and p46Shc comprise a significant portion of two of the major protein substrates phosphorylated on tyrosine in response to interleukin-2 (IL-2), IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), Steel factor (SLF), and colony-stimulating factor-1 (CSF-1). Once tyrosine phosphorylated, p52Shc and p46Shc associated with grb2. However, in contrast to published results with epidermal growth factor, treatment with GM-CSF, IL-3, and SLF failed to induce significant biochemically detectable translocation of Shc, grb2, or mSos1 from the cytosol to the plasma membrane. In addition, we did not observe significant epidermal growth factor-induced translocation of Sos1 to the membrane in Rat-1 cells. Treatment with SLF or IL-3 did increase tyrosine phosphorylation of membrane-localized p52Shc, which could then associate with grb2, although the majority of tyrosine-phosphorylated Shc was located in the cytosol. SLF, IL-3, and phorbol ester induced a decrease in the electrophoretic mobility of mSos1. This occurred with slower kinetics than p21ras activation and unlike hemopoietin-induced activation of p21ras was partially inhibited by a specific protein kinase C inhibitor. Thus, growth factor-induced modification of mSos1 may represent a downstream event, subsequent to p21ras activation. Significantly, IL-4, a cytokine that fails to activate p21ras, also failed to induce significant tyrosine phosphorylation of Shc or a shift in mSos1 mobility for the first time correlating these events with the ability of a growth factor to activate p21ras. Together, these data suggest that the current model for regulation of p21ras, which proposes a stable association of Shc-grb2-Sos1 complexes at the plasma membrane, may be an oversimplification.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Factores de Crecimiento de Célula Hematopoyética/fisiología , Interleucina-4/fisiología , Proteínas de la Membrana/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteínas/metabolismo , Animales , Transporte Biológico , Médula Ósea/metabolismo , Células de la Médula Ósea , Membrana Celular/metabolismo , Células Cultivadas , Proteína Adaptadora GRB2 , Humanos , Interleucina-2/fisiología , Interleucina-3/fisiología , Mastocitos/metabolismo , Ratones , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Adaptadoras de la Señalización Shc , Proteínas Son Of Sevenless , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Acetato de Tetradecanoilforbol/farmacología , Tirosina/metabolismoRESUMEN
The activation of erk/mitogen-activated protein kinases and p21ras is strongly associated with progression through the cell cycle. Cell growth induced by the cytokine interleukin-4 (IL-4) effectively dissociates the activation of p44erk-1 and p42erk-2 mitogen-activated protein kinases and p21ras from cell proliferation. In two cell lines of T lymphocyte and myeloid origin that were dependent upon IL-4 for continuous growth, IL-4 failed to detectably activate or induce tyrosine phosphorylation of p44erk-1 and p42erk-2. The activation of p21ras was also not detectably affected by IL-4 treatment of these cells. Treatment of the same cells with other growth factors (colony-stimulating factor-1 and Steel factor) or phorbol esters induced the tyrosine phosphorylation and activation of p44erk-1 and p42erk-2 and stimulated p21ras activity. The presence of IL-4 neither diminished nor enhanced the activation of p44erk-1 and p42erk-2 by colony-stimulating factor-1, Steel factor, or 12-O-tetradecanoylphorbol-13-acetate. Furthermore, IL-4 also failed to activate p44erk-1, p42erk-2, and p21ras in normal T lymphocytes and mast cells derived from spleen and bone marrow, respectively. Significantly, these findings demonstrate that IL-4-induced cell growth may be dissociated from the activation of p44erk-1, p42erk-2, and p21ras, suggesting that their activation may not be an absolute requirement for growth factor-stimulated mitogenesis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/fisiología , Interleucina-4/fisiología , Proteínas Quinasas Activadas por Mitógenos , Proteína Oncogénica p21(ras)/metabolismo , Médula Ósea/metabolismo , Células de la Médula Ósea , Células Cultivadas , Activación Enzimática , Humanos , Mastocitos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/metabolismo , Tirosina/metabolismoRESUMEN
p56lck, a member of the src family of non-receptor protein tyrosine kinases, is expressed almost exclusively in cells of lymphoid origin. p56lck is known to associate with the T lymphocyte surface glycoproteins CD4 and CD8, and plays a critical role in both T lymphocyte development and activation. p56lck also associates with the beta-subunit of the IL-2R, and is activated when IL-2 binds to its receptor. Using primary cultures of Con A-activated normal splenic mouse T lymphocytes, we observed an IL-2-induced sequence of events involving p56lck. We saw a rapid (within 1 to 2 min) and transient increase in p56lck kinase activity, which preceded the activation of both the p42erk-2 and p44erk-1 mitogen-activated protein kinases, maximal activation of which was observed after 10 min. We also observed an IL-2-induced shift in the electrophoretic mobility of p56lck from an apparent molecular mass of 56 kDa (p56lck) to 60 kDa (p60lck), which reached a maximum at 15 min, the level of p60lck remaining constant for up to 4 h thereafter. This IL-2-induced shift correlated with the phosphorylation of serine-59 of p56lck, a site that mitogen-activated protein kinases are capable of modifying in vitro. The implications of these results for the understanding of both p56lck function and lymphoid cell receptor signaling pathways are discussed.