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Second Messengers

CYCLICNUCLEOTIDES

cAMPandaMajorTarget, PKA

The “fight or flight” mechanism, more accurately referred to as the adrenal response, prepares the body for situations of extreme stress. Release of the hormone epinephrine, also knownasadrenaline,fromtheadrenalglandintotheblood rapidly triggers vital cellular and physiological reactions that prepare the body for intense physical activity. One of the most important effects is the breakdown of glycogen into glucose to fuel muscles (Sutherland 1972). Earl Sutherland first showed that epinephrine bound to cell-surface receptors stimulates membrane-associated adenylyl cyclases to synthesize the chemical messenger cAMP. Martin Rodbell and Alfred Gillman then discovered that G-protein subunits are the intermediates that shuttle between receptors and a family of eight adenylyl cyclase isoforms in the plasma membrane. Each G protein is a trimer consisting of Ga,Gb, and Gg subunits. The Ga subunits in the G proteins Gs and Gi are distinct and provide the specificity foractivation and inhibition of adenylyl cyclase, respectively. Gs and Gi can thus couple binding of ligands to GPCRs with either activation or inhibition of adenylyl cyclase, depending on the receptor type. This increases or reduces production of cAMP, which diffuses from the membrane into the cell. Edwin Krebs and Edmund Fischer later found that a principal task of cAMP is to stimulate protein phosphorylation (Fischer and Krebs 1955), ultimately showing cAMP-dependent protein kiPKAisthemajortargetforcAMP(seeFig.2)(SassoneCorsi 2012). It is a heterotetramer consisting of two regulatory(R)subunitsthatmaintaintwocatalytic(C)subunits in an inhibited state. When cAMP levels are low, the PKA holoenzyme is dormant; however, when cAMP levels are elevated, two molecules bind in a highly cooperative mannertoeachRsubunit,causingaconformationalchangethat releases the active C subunits (Taylor et al. 2012). In humans, four genes encode R subunits (RIa,RIb, RIIa, and RIIb)andtwogenesencodeCsubunits(CaandCb),combinations of which are expressed in most, if not all, tissues. The C subunits of PKA phosphorylate serine or threonine residues on target substrates, typically within the sequence RRxS/TF, in which F is an aliphatic hydrophobic residue oranaromaticresidue(Kempetal.1976).Around300–500 distinct intracellular proteins can be phosphorylated by PKAinatypicalcell.Theseincludeglycogenphosphorylase as part of the fight-or-flight mechanism. Active phosphorylase catalyzes the production of glucose 1-phosphate, a metabolic intermediate that is ultimately converted into other modified sugars that are used in various aspects of cellular catabolism and ATP production (see Hardie 2012). Onits release from the PKA holoenzyme, the C subunit of PKAcan diffuse into the nucleus, where it phosphorylates transcription factors such as the cAMP-response-elementbinding protein. cAMP can thereby ultimately influence transcriptional activation and reprogramming of the cell. Additional layers of regulation ensure that PKA phosphorylates the correct proteins at the right time. For example, prostaglandin E1 and epinephrine both produce similar increases in cAMP and PKA activity in the heart, but onlyepinephrine increases glycogen phosphorylase activity. This is achieved in part through attachment of PKA to subcellular structures via interactions between R subunits and A-kinase-anchoring proteins (AKAPs) (Wong and Scott 2004). Anchoring of PKA to subcellular structures by AKAPs is a means to limit the range of action of PKA and avert the indiscriminate transmission of these responses throughout the cell. A typical cell expresses 10–15 different AKAPs. These proteins all contain an amphipathic-helix motif that binds to a docking and dimerization domain formed by the R subunits of PKA and targeting domains that tether the AKAPs to intracellular membranes or organelles. AKAPs promote signaling efficacy by placing PKA near preferred substrates and insulating different anchored PKAcomplexes fromoneanother(ScottandPawson2009).MostAKAPs also organize other signaling proteins, such as GPCRs, GTPases, protein phosphatases, phosphodiesterase (PDE), and other protein kinases.