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Protein kinase A

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CAMP-dependent protein kinase
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In cell biology, protein kinase A (PKA[N 1]) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (EC 2.7.11.11). Protein kinase A has several functions in the cell, including regulation of glycogen, sugar, and lipid metabolism.

It should not be confused with AMP-activated protein kinase – which, although being of similar nature, may have opposite effects[1] – nor be confused with cyclin-dependent kinases (Cdks), nor be confused with the acid dissociation constant pKa.

Mechanism

Overview: Activation and inactivation mechanisms of PKA

Activation

The PKA enzyme is also known as cAMP-dependent enzyme because it is activated only when cAMP is present. Hormones such as glucagon and epinephrine begin the activation cascade (that triggers protein kinase A) by binding to a G protein–coupled receptor (GPCR) on the target cell. When a GPCR is activated by its extracellular ligand, a conformational change is induced in the receptor that is transmitted to an attached intracellular heterotrimeric G protein complex by protein domain dynamics. The Gs alpha subunit of the stimulated G protein complex exchanges GDP for GTP and is released from the complex. The activated Gs alpha subunit binds to and activates an enzyme called adenylyl cyclase, which, in turn, catalyzes the conversion of ATP into cyclic adenosine monophosphate (cAMP) – increasing cAMP levels. Four cAMP molecules are required to activate a single PKA enzyme. This is done by two cAMP molecules binding to each of the two regulatory subunits on a PKA enzyme causing the subunits to detach exposing the two (now activated) catalytic subunits. Next the catalytic subunits can go on to phosphorylate other proteins.[2]

Below is a list of the steps involved in PKA activation:

  1. Cytosolic cAMP increases
  2. Two cAMP molecules bind to each PKA regulatory subunit
  3. The regulatory subunits move out of the active sites of the catalytic subunits and the R2C2 complex dissociates
  4. The free catalytic subunits interact with proteins to phosphorylate Ser or Thr residues.

Catalysis

The free catalytic subunits can then catalyze the transfer of ATP terminal phosphates to protein substrates at serine, or threonine residues. This phosphorylation 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:

  • In direct protein phosphorylation, PKA directly either increases or decreases the activity of a protein.
  • In protein synthesis, PKA first directly activates CREB, which binds the cAMP response element, altering the transcription and therefore the synthesis of the protein. In general, this mechanism takes more time (hours to days).

Phosphorylation mechanism

The Serine/Threonine residue of the substrate peptide is orientated in such a way that the hydroxyl group faces towards 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 transfer of gamma-phosphate to the Serine/Threonine of the peptide substrate. There are two conserved residues, Glutamate 91 and Lysine 72, that mediate the positioning of alpha- and beta-phosphate groups. The hydroxyl group 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 phosphodiesterbond between the beta-phosphate and the gamma-phosphate groups.

Inactivation

cAMP

Downregulation of protein kinase A occurs by a feedback mechanism using phosphodiesterase, which is one of the substrates activated by the kinase. Phosphodiesterase quickly converts cAMP to AMP, thus reducing the amount of cAMP that can activate protein kinase A.

Thus, PKA is controlled by cAMP. Also, the catalytic subunit itself can be down-regulated by phosphorylation.

Anchorage

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 of A-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.

Function

PKA phosphorylates proteins that have the motif Arginine-Arginine-X-Serine exposed, in turn (de)activating the proteins. 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 with cell type:

Overview table

Cell type Organ/system Stimulators
ligands --> Gs-GPCRs
or PDE inhibitors 		Inhibitors
ligands --> Gi-GPCRs
or PDE stimulators 		Effects
adipocyte
myocyte (skeletal muscle) muscular system
myocyte (cardiac muscle) cardiovascular
myocyte (smooth muscle) cardiovascular Vasodilation
hepatocyte liver
neurons in nucleus accumbens nervous system dopamine --> dopamine receptor Activate reward system
principal cells in kidney kidney
Thick ascending limb cell kidney Vasopressin --> V2 receptor stimulate Na-K-2Cl symporter (perhaps only minor effect)[5]
Cortical collecting tubule cell kidney Vasopressin --> V2 receptor stimulate Epithelial sodium channel (perhaps only minor effect)[5]
Inner medullary collecting duct cell kidney Vasopressin --> V2 receptor
proximal convoluted tubule cell kidney PTH --> PTH receptor 1 Inhibit NHE3 --> ↓H+ secretion[7]
juxtaglomerular cell kidney renin secretion

In adipocytes and hepatocytes

Adrenaline and glucagon affect the activity of protein kinase A by changing the levels of cAMP in a cell via the G-protein mechanism, using adenylate cyclase. Protein kinase A acts to phosphorylate many enzymes important in metabolism. For example, protein kinase A phosphorylates acetyl-CoA carboxylase and pyruvate dehydrogenase. Such covalent modification has an inhibitory effect on these enzymes, thus inhibiting lipogenesis and promoting net gluconeogenesis. 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.

In nucleus accumbens neurons

PKA helps transfer/translate the dopamine signal into cells in the nucleus accumbens, which mediates reward, motivation, and task salience. The vast majority of reward perception involves neuronal activation in the nucleus accumbens, some examples of which include sex, recreational drugs, and food.[citation needed]

See also

References

  1. ^ Hallows KR, Alzamora R, Li H, et al. (April 2009). "AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells". Am. J. Physiol., Cell Physiol. 296 (4): C672–81. doi:10.1152/ajpcell.00004.2009. PMC 2670645. PMID 19211918.
  2. ^ Voet, Voet & Pratt (2006). Fundamentals of Biochemistry. Wiley. Pg 492
  3. ^ a b c d e Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. ISBN 0-443-07145-4. Page 172
  4. ^ Rodriguez P, Kranias EG. (December 2005). "Phospholamban: a key determinant of cardiac function and dysfunction". Arch Mal Coeur Vaiss. 98 (12): 1239–43. PMID 16435604.
  5. ^ a b c d e Walter F., PhD. Boron. Medical Physiology: A Cellular And Molecular Approaoch. Elsevier/Saunders. ISBN 1-4160-2328-3. Page 842
  6. ^ Walter F., PhD. Boron. Medical Physiology: A Cellular And Molecular Approaoch. Elsevier/Saunders. ISBN 1-4160-2328-3. Page 844
  7. ^ Walter F., PhD. Boron. Medical Physiology: A Cellular And Molecular Approaoch. Elsevier/Saunders. ISBN 1-4160-2328-3. Page 852
  8. ^ a b c d Walter F., PhD. Boron (2003). Medical Physiology: A Cellular And Molecular Approaoch. Elsevier/Saunders. p. 1300. ISBN 1-4160-2328-3. Page 867

Notes

  1. ^ Not to be confused with pKa, the symbol for the acid dissociation constant.
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