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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 "Similar 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

 

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References