RESUMEN
The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Adenilil Ciclasas/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Albuterol/farmacología , Animales , Canales de Calcio Tipo L/genética , Línea Celular , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Conductividad Eléctrica , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Isoproterenol/farmacología , Cinética , Sustancias Macromoleculares , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Pruebas de Precipitina , Prosencéfalo/citología , Prosencéfalo/metabolismo , Unión Proteica , Células Piramidales/citología , Células Piramidales/efectos de los fármacos , Células Piramidales/enzimología , Células Piramidales/metabolismo , Ratas , Receptores Adrenérgicos beta 2/genética , Especificidad por SustratoRESUMEN
To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.
Asunto(s)
Transformación Celular Neoplásica/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Janus Quinasa 3/genética , Leucemia/genética , ARN Interferente Pequeño/farmacología , Animales , Sitios de Unión , Línea Celular Tumoral , Humanos , Subunidad gamma Común de Receptores de Interleucina/antagonistas & inhibidores , Subunidad gamma Común de Receptores de Interleucina/genética , Janus Quinasa 3/metabolismo , Leucemia/metabolismo , Leucemia/patología , Ratones , Datos de Secuencia Molecular , Mutación , Fosforilación , Transducción de SeñalRESUMEN
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Aminoácidos/química , Animales , Encéfalo/metabolismo , Dominio Catalítico , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Concentración 50 Inhibidora , Ionóforos/farmacología , Microcistinas , Ácido Ocadaico/farmacología , Péptidos Cíclicos/farmacología , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 2 , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Serina/química , Treonina/químicaRESUMEN
Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
SAP97, a PDZ-containing protein, is reported to concentrate in axon terminals, where its function remains unknown. Using highly specific new antibodies, we show that SAP97 in rat cerebral cortex is associated with heteromeric AMPA receptors via a selective biochemical interaction between SAP97 and the GluR1 subunit. Using light and electron microscopic immunocytochemistry, we demonstrate cellular and synaptic colocalization of SAP97 and GluR1, and show that SAP97 concentrates at synapses that contain GluR1 but not necessarily GluR2 or GluR3. Using quantitative postembedding immunogold electron microscopy, we find that SAP97 is at highest concentration within the postsynaptic density of asymmetric synapses. These data suggest that SAP97 may help to anchor GluR1-containing AMPA receptors at the synapse. As a multifunctional scaffolding protein, SAP97 may organize components of AMPA-related intracellular signalling pathways, including those associated with calcium-permeable homomeric GluR1 channels.
Asunto(s)
Corteza Cerebral/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Membranas Sinápticas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Corteza Cerebral/ultraestructura , Masculino , Proteínas de la Membrana , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Membranas Sinápticas/ultraestructuraRESUMEN
Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.