RESUMEN
Since the cloning of dual-specificity A kinase-anchoring protein 2 (D-AKAP2), there has been considerable progress in understanding the structural features of this AKAP and its interaction with protein kinase A (PKA). The domain organization of D-AKAP2 is quite unique, containing two tandem, putative RGS domains, a PKA-binding motif, and a PDZ (PSD95/Dlg/ZO1)-binding motif. Although the function of D-AKAP2 has remained elusive, several reports suggest that D-AKAP2 is targeted to cotransporters in the kidney and that it may play a role in regulating transporter activity. In addition, the finding that a single nucleotide polymorphism in the PKA-binding region of D-AKAP2 may contribute to increased morbidity and mortality emphasizes the potential importance of this protein in pathogenesis. The first part of this article focuses on initial efforts to identify and clone D-AKAP2, followed by tissue localization and expression profiles. The latter half of the article focuses on the domain organization of D-AKAP2 and its interaction with PKA. Finally, a comprehensive analysis of the PKA binding motif is described, which has led to the development of novel peptides derived from D-AKAP2 that can be useful tools in probing the function of this AKAP in cellular and animal models.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Anclaje a la Quinasa A , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Deuterio/metabolismo , Humanos , Isoenzimas/metabolismo , Espectrometría de Masas/métodos , Mitocondrias/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIalpha in addition to the RII subunits. RESULTS: Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIalpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIalpha and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIalpha and RIIbeta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCbeta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. CONCLUSIONS: The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIalpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIalpha in these regions. Likewise, gravin may be an anchor of PKCbeta at the NMJ.