PKA & PKG

Cyclic AMP-dependent protein kinase (PKA or cAK) and cyclic GMP-dependent protein kinase (PKG or cGK) transfer the γ-phosphate of ATP to serine and threonine residues of many cellular proteins. PKAs are present in most cells and function as effectors of many cAMP-elevating first messengers such as hormones and neurotransmitters. PKG is highly expressed in special cell types such as smooth muscle cells, platelets and cerebellar Purkinje cells (PKG I), as well as intestinal, kidney and brain cells (PKG II). cGMP-elevating agents include NO, natriuretic peptides and guanylin. In most tissues, PKGs are much less abundantly expressed than PKAs.

In the absence of its activating ligand cAMP, PKA exists as an inactive holoenzyme of two regulatory (R) and two catalytic (C) subunits. Following an increase in intracellular cAMP, the R-subunits bind cAMP resulting in the dissociation of the holoenzyme and the release of two free active C-subunits. Active C-subunit phosphorylates peptide substrates containing the -R-R/K-X-S/T- substrate consensus amino acid sequence (although exceptions to this consensus sequence have been observed). The PKA type I holoenzymes (RIα2C2, RIβ2C2) are predominantly cytoplasmic, whereas the majority of type II PKA (RIIα2C2, RIIβ2C2) associates with cytoskeletal structures, organelles and membranes. The holoenzymes can be anchored to specific compartments via interaction of their regulatory subunits with specific PKA anchoring proteins (AKAPs - most of which are identified for PKA II so far).

In contrast to PKA, the regulatory and catalytic regions of the PKG enzyme are present in one polypeptide. Binding of cGMP to the two cGMP-binding sites is thought to release the autoinhibitory N-terminal domain from binding to the C-terminal catalytic domain, thus enabling substrate binding and heterophosphorylation. The substrate consensus amino acid sequence for PKGs appears to require more multiple basic residues than does PKA (consensus -R/K2-3-X-S/T-). However, in vitro, many substrate proteins can be phosphorylated by both kinases. In addition to phosphorylating other proteins (heterophosphorylation), each of the PKGs and type II PKA phosphorylate themselves (autophosphorylation). Within the cell, the specific localization of the kinases and their substrates has been shown to restrict some of the possible interactions suggested by in vitro data. PKG I is localized mainly in the cytoplasm and a number of PKG anchoring proteins (GKAPs, especially for the type Iα enzyme) have been identified. The PKG II enzyme is anchored to membranes via its myristoylated N-terminus.

PKA has been shown to mediate many cellular responses to the intracellular second messenger cAMP in eukaryotes. Other important effectors of cAMP are the guanine nucleotide exchange factors Epac 1 and 2, which activate Rap1, a member of the Ras family of small GTPases. cAMP is also known to directly regulate ion channels. PKA I has been shown to mediate cAMP effects on inhibition of lymphocyte cell proliferation and immune response as well as long term depression in the hippocampus and sensory nerve transmission. PKA II is involved in the regulation of neuronal gene expression and motor learning as well as lipolysis and sperm motility. However, differences between PKA I and II functions are probably mainly due to differences in levels of expression in specific cells and the relative ability of each kinase subtype to localize near substrates by binding to scaffolding proteins (AKAPs). For example PKA II can be localized via AKAPs to the Golgi-centrosomal area, to receptors and ion channels, to the cytoskeleton and the nucleus.

The second messenger cGMP has three major effector systems within the cell: cGMP-regulated ion channels, cGMP-regulated phosphodiesterases and PKGs. PKG I mediates cGMP-induced smooth muscle cell relaxation and inhibition of platelet aggregation. These effects correlate at least partly with an inhibition of calcium release from intracellular stores. In addition PKG I can inhibit cardiac myocyte contractility and has also been shown to regulate proliferation and gene expression in various cell types. PKG II stimulates intestinal chloride secretion, inhibits renin release from juxtaglomerular cells, stimulates renal calcium reabsorption and regulates endochondral ossification.

The Table below contains accepted modulators and additional information. For a list of additional products, see the "Products" section below.

Family Members	PKA I (RI2C2) and PKA II (RII2C2) (P5511 (b))	PKA I (RI2C2) and PKA II (RII2C2) (P5511 (b))	PKA I (RI2C2) and PKA II (RII2C2) (P5511 (b))
Other Names	cAK I and cAK II	cAK I and cAK II	cAK I and cAK II
Molecular Weight (kDa)	43-47 kDa (R I)	49-55 kDa (R II)	40 kDa (C)
Structural Data	Tetramer of 2 regulatory (R) and two catalytic (C) subunits R I	Tetramer of 2 regulatory (R) and two catalytic (C) subunits R II	Tetramer of 2 regulatory (R) and two catalytic (C) subunits C (P2645 (b), P6998 (h), C8482 (h))
Isoforms	RIα: 381 aa RIβ: 381 aa	RIIα: 404 aa RIIβ: 418 aa	Cα: 351 aa Cβ: 351 aa Cγ: 351 aa
Species	Human	Human	Human
Domain Organization	N-terminal dimerization/AKAP-binding and autoinhibitory region, followed by two cAMP binding domains (similar organization in RI and RII)	N-terminal dimerization/AKAP-binding and autoinhibitory region, followed by two cAMP binding domains (similar organization in RI and RII)	Kinase domain
Phosphorylation Sites	Not Known	Ser99 (RIIα), autophosphorylation Thr54 (RIIα), phosphorylated by CDK1 Ser114 (RIIβ), autophosphorylation	Thr197 Ser338 (associated with activation)
Tissue Distribution	RIα: expressed in all tissues RIβ: preferentially expressed in neurons	RIIα: expressed in all tissues RIIβ: preferentially expressed in fat and neurons	Ubiquitous (Cα1, Cβ1) Brain (Cβ2,3) Testis (Cα2, Cγ) Lymphocytes (Cα1, Cβ2)
Subcellular Localization	Cytoplasm Organelles	Cytoskeletal structures Organelles Membranes	Same as R-subunits
Binding Partners/ Associated Proteins	AKAPs: D-AKAP1 AKAP220	AKAPs: MAP2 AKAP-79 AKAP95 S-AKAP84/D-AKAP1 AKAP220 AKAP450 AKAP18 WASP mAKAP Others	N-terminal myristoylation
Upstream Activators	cAMP	cAMP	Not Known
Downstream Activation	Not Known	Not Known	Multiple substrates
Activators	cAMP (A6885)a 8-Br-cAMP (B7880)a 6-Bnz-cAMP (B4560)b 6-MB-cAMP	cAMP (A6885)a Sp-5,6-DCl-cBIMPS (S6069)a 6-Bnz-cAMP (B4560)b	See R-subunits
Inhibitors	Rp-8-Br-cAMPS (B2432) Rp-8-Cl-cAMPS (C0610) Rp-cAMPS (A165)	Rp-8-pCPT-cAMPS (C0735) Rp-cAMPS (A165)	PKI (P0300) H89 (B1427) H8 (M9656) KT5720 (K3761)
Isozyme Selective Activators	6-Bnz-cAMP (B4560) or 8-PIP-cAMP (P0872) in combination with 8-AHA-cAMP or 8-MA-cAMP	6-Bnz-cAMP (B4560) or 6-Phe-cAMP in combination with Sp-5,6-DCl-cBIMPS or Sp-8-pCPT-camps	Not Known
Physiological Function	Inhibition of T-cell activation (RIα) Nociceptive processing in the spinal cord (RIβ) Long-term depression in the hippocampus (RIβ) Mesoderm development (RIα)	Lipolysis (RIIβ) Increased myocardial contractility Inhibition of T-cell activation Long-term depression in the visual cortex (RIIβ) Motor learning (RIIβ) Neuronal gene expression (RIIβ)	Gene expression Growth and differentiation Cytoskeleton dynamics Ion channel conductivity Sperm motility (Cα2)
Disease Relevance	Carney complex (RIα)	Diabetes (RIIβ)	See R-subunits

Family Members	PKG I	PKG II
Other Names	cGK I	cGK II
Molecular Weight (kDa)	74-80 kDa	86 kDa
Structural Data	Dimer	Dimer
Isoforms	PKG Iα: 671 aa (P3488 (b)) PKG Iβ: 686 aa (P3738 (h))	PKG II: 762 aa (P2363 (rat))
Species	Human	Human
Domain Organization	N-terminal regulatory domain containing dimerization/GKAP-binding and auto- inhibitory sites, two central cGMP binding domains, C-terminal kinase domain	N-terminal regulatory domain containing dimerization/GKAP-binding and auto- inhibitory sites, two central cGMP binding domains, C-terminal kinase domain
Phosphorylation Sites	Ser51 Thr59 Ser65 Ser73 (PKG Iα), autophosphorylation Ser64 Ser80 (PKG Iβ), autophosphorylation	Ser110 Ser114 Ser126 Ser445, autophosphorylation
Tissue Distribution	All types of smooth muscle Platelets (Iβ) Cerebellar Purkinje cells Lung (Iα) Lymphocytes (Iβ) Cardiac myocytes (Iα) Endothelial cells (not all)	Intestinal mucosa Kidney Adrenal cortex Brain Bone (chondrocytes) Lung (Clara cells)
Subcellular Localization	Cytoplasm Cytoskeletal structures	Membranes
Binding Partners/ Associated Proteins	GKAPs: MBS (Iα) Troponin T (T0175) GKAP-42 (Iα) IRAG (Iβ) RGS-2 (Iα)	N-terminal myristoylation NHERF2
Upstream Activators	cGMP	cGMP
Downstream Activation	Many substrates, e.g. VASP, IRAG, BKCa channel, MBS, PDE5, G-substrate	Substrates, e.g. CFTR
Activators	cGMP (G6129) PET-cGMP 8-Br-cGMP (B1381) 8-pCPT-cGMP (C5438)	cGMP (G6129) 8-pCPT-cGMP (C5438) 8-Br-cGMP (B1381) PET-cGMP (P0622)
Inhibitors	Rp-8-Br-PET-cGMPS (B6684) Rp-8-pCPT-cGMPS (C240) KT5823 (K1388) (in vitro only) DT-2 DT-3 (peptide-based)	Rp-8-pCPT-cGMPS (C240) Rp-8-Br-PET-cGMPS (B6684)
Isozyme Selective Activators	Not Known	Not Known
Physiological Function	Smooth muscle relaxation (e.g. vasodilation, penile erection) Inhibition of platelet aggregation Facilitation of nociceptive transmission in the spinal cord Inhibition of T-cell proliferation Negative inotropic and antihypertrophic effect in heart Regulation of gene expression	Cl- secretion and inhibition of Na+ reabsorption (small intestine) Inhibition of renin secretion Stimulation of renal Ca2+ reabsorption Stimulation of aldosterone production Progression of circadian clock Endochondral ossification
Disease Relevance	Atherosclerosis	STa-induced diarrhea

Footnotes

a) cAMP and most cAMP analogs also activate Epac. To distinguish PKA and Epac effects in intact cells the Epac-specific activators 8-pCPT-2’-O-Me-cAMP or 8-Br-2’-O-Me-cAMP can be used.

b) 6-modified cAMP analogs have a higher affinity for PKA over Epac.

Abbreviations

AHA: Aminohexyl amino
8-AHA-cAMP: 8-(6-Aminohexyl)aminoadenosine-3’,5’-cyclicmonophosphate
AKAP: PKA-anchoring protein
BKCa: Ca2+-regulated potassium channel
Bnz: Benzoyl
6-Bnz-cAMP: N6-Benzoyladenosine-3’,5’-cyclic monophosphate
Br: Bromo
cAMP: Adenosine-3’, 5’-cyclic monophosphate
cAMPS: Adenosine-3’, 5’-cyclic monophosphorothioate
CFTR: Cystic fibrosis transmembrane conductance regulator
cGMP: Guanosine-3’, 5’-cyclic monophosphate
cGMPS: Guanosine-3’, 5’-cyclic monophosphorothioate
CPT: Chlorophenylthio
Epac: Exchange protein directly activated by cAMP
GKAP: PKG-anchoring protein
H8: N-(2-[Methylamino]ethyl)-5-isoquinoline-sulfonamide
H89: N-(2-[p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide
IRAG: IP3-receptor-associated cGMP kinase substrate
KT5720: (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3’,2’,1’-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester
KT5823: (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3’,2’,1’-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester
MA: Methylamino
8-MA-cAMP: 8-Methylaminoadenosine-3’,5’-cyclic monophosphate
MB: Monobutyryladenosine
MAP: Microtubule-associated protein
MBS: Myosin-binding subunit of myosin light chain phosphatase
NHERF2: Na+/H+ exchanger regulatory factor 2
PET: β-Phenyl-1,N2-ethenol
PET-cGMP: β-Phenyl-1,N2-ethenoguanosine 3’,5’-monophosphate
Phe: Phenyladenosine
PIP: Piperidino
8-PIP-cAMP: 8-Piperinoadenosine 3’,5’-cyclic monophosphate
PKA: cAMP-dependent protein kinase
PKG: cGMP-dependent protein kinase
PKI: cAMP-dependent protein kinase inhibitor peptide
RGS-2: Regulator of G-protein signaling-2
Rp-8-Br-cAMPS: 8-Bromoadenosine-3’,5’-cyclic monophosphorothioate, Rp isomer
Rp-8-Cl-cAMPS: 8-Chloroadenosine-3’,5’-cyclic monophosphorothioate, Rp isomer
Rp-cAMPS: Adenosine 3’,5’-cyclic monophosphothioate, Rp-isomer
Rp-8-pCPT-cAMPS: 8-(4-Chlorophenylthio)adenosine-3’,5’-cyclic monophosphorothioate, Rp isomer
Rp-8-Br-PET-cGMPS: b-Phenyl-1,N2-etheno-8-bromoguanosine-3’,5’-cyclic monophosphorothioate, Rp isomer
Rp-8-pCPT-cGMPS: 8-(4-Chlorophenylthio)guanosine-3’,5’-cyclic monophosphorothioate, Rp isomer
Sp-5,6-DCl-cBIMPS: 5, 6-Dichloro-1-b-D-ribofuranosylbenzimidazole-3’, 5’-cyclic monophosphorothioate, Sp-isomer
Sp-8-pCPT-cAMPS: 8-(4-Chlorophenylthio)adenosine-3’,5’-cyclic monophosphorothioate, Sp-isomer
STa: heat-stable enterotoxin secreted by enteropathogenic bacteria
VASP: vasodilator-stimulated phosphoprotein
WASP: Wiskott-Aldrich syndrome protein

References

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