Pharmacological PKA Inhibition : All May Not Be What It Seems

Page 1 Overview of cAMP Signaling The transduction of extracellular signals to intracellular responses is one of the most important and complicated aspects of cellular life. The cyclic adenosine monophosphate (cAMP) signaling pathway is involved in numerous processes and is widely regarded as the “classical” second messenger signaling pathway. cAMP is synthesized from adenosine triphosphate (ATP) by adenylyl cyclase and is broken down to 5′ AMP by a class of proteins known as phosphodiesterases (PDEs) (1, 2). Various stimuli activate adenylyl cyclase, but the best studied is ligand occupation of heterotrimeric guanine nucleotide-binding protein (G protein)–coupled receptors (GPCRs) coupled to Gs. Agonist occupation of Gs-coupled receptors catalyzes the exchange of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) on the α subunit of the G protein, causing a conformational change and dissociation of this complex from the βγ subunits. The α subunit can then interact with and activate adenylyl cyclases (Fig. 1). Receptors coupled to a different G protein, Gi, cause down-regulation of adenylyl cyclase activity and consequent lowering of cAMP concentrations (Fig. 1). cAMP has three direct intracellular targets: protein kinase A (PKA), the exchange protein activated by cAMP (Epac), and cyclic nucleotide–gated ion channels (CNGCs). CNGCs, nonselective cation channels that open upon cyclic nucleotide binding, are particularly important in the olfactory and visual systems (3). Epac is a guanine nucleotide exchange factor for the small G protein Rap1 and has been implicated in a number of cellular processes such as insulin secretion, neurotransmitter release, and integrin-mediated cell adhesion (4–6). By far the best-studied aspect of cAMP signaling, though, involves cAMP-mediated activation of PKA. Protein kinase A. PKA was discovered in the laboratory of Edwin G. Krebs in the 1960s (7). Since then it has been implicated in numerous cellular processes, including modulation of other protein kinases, regulation of intracellular calcium concentration, and regulation of transcription [reviewed in (8)]. Transcriptional responses to increased cAMP occur through activation of the cAMP response element–binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor 1 (ATF1) (9). Each of these transcription factors contains a kinaseinducible domain containing a conserved site for phosphorylation by PKA. In its inactive state, PKA exists as a tetramer consisting of two regulatory and two catalytic subunits (Fig. 2). Four molecules of cAMP bind to the regulatory subunits to activate PKA, with two cAMPbinding sites, termed the A and B sites, that are present on each regulatory subunit. cAMP binding promotes a conformational change in PKA that initiates the dissociation of the catalytic subunits, leaving a dimer of the two regulatory subunits with four bound cAMP molecules. The two PKA catalytic monomers bind ATP; they then become catalytically active and can phosphorylate serine and threonine residues on proteins containing the appropriate substrate sequence (1) (Fig. 2). PKA signaling can occur in a very small defined domain because of the anchoring of PKA near its targets by A-kinase anchoring proteins (AKAPs), which tether PKA to particular cellular organelles and to the plasma membrane (10). PKA can also activate phosphodiesterases and promote cAMP breakdown in a negative feedback mechanism (2).