RESUMEN
Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Isoenzimas/metabolismo , Miocitos Cardíacos/enzimología , Transducción de Señal , Proteínas de Anclaje a la Quinasa A/genética , Adenilil Ciclasas/genética , Animales , Células Cultivadas , Isoenzimas/genética , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
A-kinase anchoring proteins (AKAPs) influence the spatial and temporal regulation of cAMP signaling events. Anchoring of PKA in proximity to certain adenylyl cyclase (AC) isoforms is thought to enhance the phosphorylation dependent termination of cAMP synthesis. Using a combination of immunoprecipitation and enzymological approaches, we show that the plasma membrane targeted anchoring protein AKAP9/Yotiao displays unique specificity for interaction and the regulation of a variety of AC isoforms. Yotiao inhibits AC 2 and 3, but has no effect on AC 1 or 9, serving purely as a scaffold for these latter isoforms. Thus, Yotiao represents an inhibitor of AC2. The N terminus of AC2 (AC2-NT), which binds directly to amino acids 808-957 of Yotiao, mediates this interaction. Additionally, AC2-NT and Yotiao (808-957) are able to effectively inhibit the association of AC2 with Yotiao and, thus, reverse the inhibition of AC2 by Yotiao in membranes. Finally, disruption of Yotiao-AC interactions gives rise to a 40% increase in brain AC activity, indicating that this anchoring protein functions to directly regulate cAMP production in the brain.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Adenilil Ciclasas/metabolismo , Encéfalo/enzimología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/genética , Inhibidores de Adenilato Ciclasa , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Línea Celular , Membrana Celular/enzimología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/metabolismo , Unión Proteica , RatasRESUMEN
We have previously used cyclic nucleotide-gated (CNG) channels as sensors to measure cAMP signals in human embryonic kidney (HEK)-293 cells. We found that prostaglandin E(1) (PGE(1)) triggered transient increases in cAMP concentration near the plasma membrane, whereas total cAMP levels rose to a steady plateau over the same time course. In addition, we presented evidence that the decline in the near-membrane cAMP levels was due primarily to a PGE(1)-induced stimulation of phosphodiesterase (PDE) activity, and that the differences between near-membrane and total cAMP levels were largely due to diffusional barriers and differential PDE activity. Here, we examine the mechanisms regulating transient, near-membrane cAMP signals. We observed that 5-min stimulation of HEK-293 cells with prostaglandins triggered a two- to threefold increase in PDE4 activity. Extracellular application of H89 (a PKA inhibitor) inhibited stimulation of PDE4 activity. Similarly, when we used CNG channels to monitor cAMP signals we found that both extracellular and intracellular (via the whole-cell patch pipette) application of H89, or the highly selective PKA inhibitor, PKI, prevented the decline in prostaglandin-induced responses. Following pretreatment with rolipram (a PDE4 inhibitor), H89 had little or no effect on near-membrane or total cAMP levels. Furthermore, disrupting the subcellular localization of PKA with the A-kinase anchoring protein (AKAP) disruptor Ht31 prevented the decline in the transient response. Based on these data we developed a plausible kinetic model that describes prostaglandin-induced cAMP signals. This model has allowed us to quantitatively demonstrate the importance of PKA-mediated stimulation of PDE4 activity in shaping near-membrane cAMP signals.
Asunto(s)
Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Prostaglandinas/farmacología , Transducción de Señal , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Adenilil Ciclasas/metabolismo , Tampones (Química) , Línea Celular , Simulación por Computador , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Humanos , Activación del Canal Iónico , Canales Iónicos/metabolismo , Isoquinolinas/farmacología , Cinética , Modelos Biológicos , Nucleótidos Cíclicos/metabolismo , Péptidos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Rolipram/farmacología , Sulfonamidas/farmacologíaRESUMEN
Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.