| cAMP-dependent protein kinase (Protein kinase A) | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 cAMP-dependent protein kinase heteroduodecamer, Sus scrofa | |||||||||
| Identifiers | |||||||||
| EC no. | 2.7.11.11 | ||||||||
| CAS no. | 142008-29-5 | ||||||||
| Alt. names | STK22, PKA, PKA C | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| |||||||||
Incell biology,protein kinase A (PKA) is a family ofserine-threonine kinases[1] whose activity is dependent on cellular levels ofcyclic AMP (cAMP). PKA is also known ascAMP-dependent protein kinase (EC2.7.11.11). PKA has several functions in the cell, including regulation ofglycogen,sugar, andlipidmetabolism. It should not be confused with5'-AMP-activated protein kinase (AMP-activated protein kinase).
Protein kinase A, more precisely known as adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase, abbreviated to PKA, was discovered by chemistsEdmond H. Fischer andEdwin G. Krebs in 1968. They won theNobel Prize in Physiology or Medicine in 1992 for their work on phosphorylation and dephosphorylation and how it relates to PKA activity.[2]
PKA is one of the most widely researchedprotein kinases, in part because of its uniqueness; out of 540 different protein kinase genes that make up the humankinome, only one other protein kinase,casein kinase 2, is known to exist in a physiological tetrameric complex, meaning it consists of four subunits.[1]
The diversity of mammalian PKA subunits was realized after Dr. Stan McKnight and others identified four possible catalytic subunit genes and four regulatory subunit genes. In 1991,Susan Taylor and colleagues crystallized the PKA Cα subunit, which revealed the bi-lobe structure of the protein kinase core for the very first time, providing a blueprint for all the other protein kinases in a genome (the kinome).[3]
When inactive, the PKA apoenzyme exists as a tetramer which consists of two regulatorysubunits and two catalytic subunits. The catalytic subunit contains the active site, a series of canonical residues found inprotein kinases that bind and hydrolyseATP, and a domain to bind the regulatory subunit. The regulatory subunit has domains to bind to cyclic AMP, a domain that interacts with catalytic subunit, and an auto inhibitory domain. There are two major forms of regulatory subunit; RI and RII.[4]
Mammalian cells have at least two types of PKAs: type I is mainly in thecytosol, whereas type II is bound via its regulatory subunits and special anchoring proteins, described in theanchorage section, to theplasma membrane,nuclear membrane,mitochondrial outer membrane, andmicrotubules. In both types, once the catalytic subunits are freed and active, they can migrate into thenucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm.[5]
The following human genes encode PKA subunits:
PKA is also commonly known as cAMP-dependent protein kinase, because it has traditionally been thought to be activated through release of the catalytic subunits when levels of thesecond messenger calledcyclic adenosine monophosphate, or cAMP, rise in response to a variety of signals. However, recent studies evaluating the intact holoenzyme complexes, including regulatory AKAP-bound signalling complexes, have suggested that the local sub cellular activation of the catalytic activity of PKA might proceed without physical separation of the regulatory and catalytic components, especially at physiological concentrations of cAMP.[6][7] In contrast, experimentally induced supra physiological concentrations of cAMP, meaning higher than normally observed in cells, are able to cause separation of the holoenzymes, and release of the catalytic subunits.[6]
Extracellular hormones, such asglucagon andepinephrine, begin an intracellular signalling cascade that triggers protein kinase A activation by first binding to aG protein–coupled receptor (GPCR) on the target cell. When a GPCR is activated by its extracellular ligand, aconformational change is induced in the receptor that is transmitted to an attached intracellularheterotrimeric G protein complex byprotein domain dynamics. TheGs alpha subunit of the stimulated G protein complex exchangesGDP forGTP in a reaction catalyzed by the GPCR and is released from the complex. The activated Gs alpha subunit binds to and activates an enzyme calledadenylyl cyclase, which, in turn, catalyzes the conversion ofATP into cAMP, directly increasing the cAMP level. Four cAMP molecules are able to bind to the two regulatory subunits. This is done by two cAMP molecules binding to each of the two cAMP binding sites (CNB-B and CNB-A) which induces a conformational change in the regulatory subunits of PKA, causing the subunits to detach and unleash the two, now activated, catalytic subunits.[8]
Once released from inhibitory regulatory subunit, the catalytic subunits can go on tophosphorylate a number of other proteins in the minimal substrate context Arg-Arg-X-Ser/Thr.,[9] although they are still subject to other layers of regulation, including modulation by the heat stable pseudosubstrate inhibitor of PKA, termed PKI.[7][10]
Below is a list of the steps involved in PKA activation:
The liberated catalytic subunits can then catalyze the transfer of ATP terminal phosphates toproteinsubstrates atserine, orthreonineresidues. Thisphosphorylation usually results in a change in activity of the substrate. Since PKAs are present in a variety of cells and act on different substrates, PKA regulation and cAMP regulation are involved in many different pathways.
The mechanisms of further effects may be divided into direct protein phosphorylation and protein synthesis:
The Serine/Threonine residue of the substrate peptide is orientated in such a way that the hydroxyl group faces the gamma phosphate group of the bound ATP molecule. Both the substrate, ATP, and two Mg2+ ions form intensive contacts with the catalytic subunit of PKA. In the active conformation, the C helix packs against the N-terminal lobe and the Aspartate residue of the conserved DFG motif chelates the Mg2+ ions, assisting in positioning the ATP substrate. The triphosphate group of ATP points out of the adenosine pocket for the transfer of gamma-phosphate to the Serine/Threonine of the peptide substrate. There are several conserved residues, include Glutamate (E) 91 and Lysine (K) 72, that mediate the positioning of alpha- and beta-phosphate groups. The hydroxyl group of the peptide substrate's Serine/Threonine attacks the gamma phosphate group at the phosphorus via an SN2 nucleophilic reaction, which results in the transfer of the terminal phosphate to the peptide substrate and cleavage of the phosphodiester bond between the beta-phosphate and the gamma-phosphate groups. PKA acts as a model for understandingprotein kinase biology, with the position of the conserved residues helping to distinguish the activeprotein kinase and inactivepseudokinase members of the human kinome.
Downregulation of protein kinase A occurs by a feedback mechanism and uses a number of cAMP hydrolyzingphosphodiesterase (PDE) enzymes, which belong to the substrates activated by PKA. Phosphodiesterase quickly converts cAMP to AMP, thus reducing the amount of cAMP that can activate protein kinase A. PKA is also regulated by a complex series of phosphorylation events, which can include modification by autophosphorylation and phosphorylation by regulatory kinases, such as PDK1.[7]
Thus, PKA is controlled, in part, by the levels ofcAMP. Also, the catalytic subunit itself can be down-regulated by phosphorylation.
The regulatory subunit dimer of PKA is important for localizing the kinase inside the cell. The dimerization and docking (D/D) domain of the dimer binds to the A-kinase binding (AKB) domain ofA-kinase anchor protein (AKAP). The AKAPs localize PKA to various locations (e.g., plasma membrane, mitochondria, etc.) within the cell.
AKAPs bind many other signaling proteins, creating a very efficient signaling hub at a certain location within the cell. For example, an AKAP located near the nucleus of a heart muscle cell would bind both PKA and phosphodiesterase (hydrolyzes cAMP), which allows the cell to limit the productivity of PKA, since the catalytic subunit is activated once cAMP binds to the regulatory subunits.
PKA phosphorylatesproteins that have the motif Arginine-Arginine-X-Serine exposed, in turn (de)activating the proteins. Many possible substrates of PKA exist; a list of such substrates is available and maintained by theNIH.[11]
As protein expression varies from cell type to cell type, the proteins that are available for phosphorylation will depend upon the cell in which PKA is present. Thus, the effects of PKA activation vary withcell type:
Epinephrine andglucagon affect the activity of protein kinase A by changing the levels of cAMP in a cell via the G-protein mechanism, usingadenylate cyclase. Protein kinase A acts to phosphorylate many enzymes important in metabolism. For example, protein kinase A phosphorylatesacetyl-CoA carboxylase andpyruvate dehydrogenase. Such covalent modification has an inhibitory effect on these enzymes, thus inhibitinglipogenesis and promoting netgluconeogenesis. Insulin, on the other hand, decreases the level of phosphorylation of these enzymes, which instead promotes lipogenesis. Recall that gluconeogenesis does not occur in myocytes.
PKA helps transfer/translate thedopamine signal into cells in thenucleus accumbens, which mediates reward, motivation, andtask salience. The vast majority of reward perception involves neuronal activation in the nucleus accumbens, some examples of which include sex, recreational drugs, and food. Protein Kinase A signal transduction pathway helps in modulation of ethanol consumption and its sedative effects. A mouse study reports that mice with genetically reduced cAMP-PKA signalling results into less consumption of ethanol and are more sensitive to its sedative effects.[18]
PKA is directed to specific sub-cellular locations after tethering toAKAPs.Ryanodine receptor (RyR) co-localizes with the muscle AKAP and RyR phosphorylation and efflux of Ca2+ is increased by localization of PKA at RyR by AKAPs.[19]
In a cascade mediated by aGPCR known asβ1 adrenoceptor, activated bycatecholamines (notablynorepinephrine), PKA gets activated and phosphorylates numerous targets, namely:L-type calcium channels,phospholamban,troponin I,myosin binding protein C, andpotassium channels. This increasesinotropy as well aslusitropy, increasing contraction force as well as enabling the muscles to relax faster.[20][21]
PKA has always been considered important in formation of amemory. In thefruit fly, reductions in expression activity of DCO (PKA catalytic subunit encoding gene) can cause severe learning disabilities, middle term memory and short term memory. Long term memory is dependent on the CREB transcription factor, regulated by PKA. A study done on drosophila reported that an increase in PKA activity can affect short term memory. However, a decrease in PKA activity by 24% inhibited learning abilities and a decrease by 16% affected both learning ability and memory retention. Formation of a normal memory is highly sensitive to PKA levels.[22]
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