The protein kinase A (PKA ) is a cAMP -dependent protein kinase and belongs to the serine / threonine kinases. It is best examined in comparison to other protein kinases and characterized. PKA (glycogen, lipids, carbohydrates) involved in the regulation in energy metabolism. PKA also plays an important role in the modification of synapses, and the control of ion channels. Finally, specific transcription factors are activated via PKA, the so-called cAMP - responsive element - binding proteins ( CREB ). Through a coactivator ( CREB -binding protein ) transcription of target genes is possible, which have a cAMP response element (cAMP responsive element ).
The name derives from the activation by cyclic AMP (cAMP ), a second messenger whose intracellular formation is triggered by a number of hydrophilic hormones.
In higher eukaryotes, the enzyme is developed in the absence of cAMP exists as a tetramer composed of two different protein subunits. Thus, it is a so-called heterotetramer ( R2C2 ). The complex consists of
- Two regulatory subunits R with high affinity for cAMP with a mass of 43-45 kDa each and
- Two catalytic subunits C and binding sites for substrate and coenzyme (ATP) with a mass of 40 kDa.
In falciparum lower, unicellular eukaryotes, such as yeast, in the slime mold Dictyostelium discoideum or Malariaereger Plasmodium, PKA exists as a heterodimer consisting of one each R and C subunit.
In mammals, four isoforms of the R subunit ( RIa, RIβ, RIIα and RIIβ ) and three types of the C subunit ( Cα, Cβ and C? ) Were detected. The different isoforms very many different heterotetramers can form. This contributes to the diversity and specificity Varibalität the PKA signaling pathway in the cell.
The R subunits all have a similar structure, and differ in overall length. At the N -terminus of a Dimersierungsdomäne is located, contact through which both R subunits. In addition, the so-called A - kinase anchor proteins ( AKAPs ) have the ability to be bound by this N-terminal domain of the PCA. At the C -terminus there are two cAMP - binding domains, which allow the binding of cAMP. Between the N-and C-terminus is an autoinhibitory sequence. It serves as a recognition sequence for the C subunit. Characterized the substrate binding site of the C- subunit is blocked until the C subunit of cAMP upon activation will be released.
The N- terminus of the C subunit has been modified with myristic acid. The significance of this is not yet understood.
Activation and effects
In higher eukaryotes, the R2C2 complex is inactive, because the regulatory subunits to block the catalytic site of the C- subunit. The cooperative binding of two molecules of cAMP to the R subunits leads by dissociation of the complex into an R2 - complex and two active C subunits. The enzyme complex ( 10-9 mol · l -1) already activated by cAMP concentrations in the order of 10 nM. By binding of cAMP to the R subunits, a large conformational change occurs. In higher eukaryotes are bound four, in the lower two molecules of cAMP. In consequence, the affinity between the R and the C- subunit by a factor of 10,000 to 100,000 is reduced, so that the C subunits can dissociate. The free C subunits can then phosphorylate a variety of substrates, the PCA is active. These substrates at specific threonine or serine residues are phosphorylated by the γ - phosphate group of ATP is transferred. The activity of PKA extends in cytosolic substrates over several minutes. When PKA phosphorylates transcription factors in the nucleus, their duration extended by several hours.
PKA has a variety of effects both in the cytosol of a cell (inter- converting enzymes ) as well as in the core ( activation of transcription factors ). Both in the cytosol, it conveys the glycogen ( glycogenolysis ) and lipid degradation ( lipolysis).
The hydrolysis of fats (triglycerides ) is controlled by PKA -regulated lipases. Simultaneously, a pacemaker enzyme of fatty acid synthesis, acetyl-CoA carboxylase, inhibits, what the lipolytic effect of the cAMP nor increases.
The protein sequence is the target of phosphorylation by PKA, having the consensus sequence Arg-Arg- X-Ser - X, where X is preferably a small hydrophobic amino acid.
The subunits of PKA appear respectively in different forms, such as
- A RI isoform
- RII isoform;
Latter differs from the RI isoform by their Autophosphorylierbarkeit. The RII subunit can be phosphorylated at one point by the C subunit of the holoenzyme ( R2C2 ). However, this does not cancel the inactive status.
The C- subunit has two specific serine / threonine phosphorylation sites. These are threonine 197 and serine 338, the former is located in the activation loop of the C- subunit. Thr 197 is an autophosphorylation target. For the catalytic activity of the C subunit three amino acids are important: aspartate 166, lysine 168 and aspartate 171 These three amino acids are highly conserved in all Serin-/Threoninproteinkinasen receive.
The PKA is regulated not only by the amount of cAMP. It is known that it can also be regulated by their subcellular whereabouts. So associated PKA, which has a RII subunit to the cytoskeleton and the Golgi apparatus. This is mediated by a specific protein, the protein kinase A anchor protein ( protein kinase A anchor protein ).
There is also still a feedback control mechanism. This activates PKA, a cAMP phosphodiesterase (PDE; see image ( B)). The activated PDE hydrolyzes cAMP to AMP. Thus, the activity of the PCA will shut itself down or comes to a halt. This is especially useful when no extracellular signal longer present in the formation of cAMP. Under these circumstances, this second messenger to be rapidly inactivated.