RESUMO
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
Assuntos
Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteína Quinase Tipo II Dependente de AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo I Dependente de AMP Cíclico/antagonistas & inibidores , Peptídeos/química , Inibidores de Proteínas Quinases/química , Subunidades Proteicas/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Proteína Quinase Tipo I Dependente de AMP Cíclico/química , Proteína Quinase Tipo I Dependente de AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico/química , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosforilação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.