PKA (Protein Kinase-A) is a second messenger-dependent enzyme that has been implicated in a wide range of cellular processes, including transcription, metabolism, cell cycle progression and apoptosis. Known modulators of PKA activity include factors that either activate or inhibit AC (Adenylate Cyclase), resulting in an increase or decrease in cAMP (Cyclic Adenosine 3',5'-monophosphate) levels. The enzyme occurs naturally as a four-membered structure with two regulatory (R) and two catalytic (C) subunits. Four genes encode the R subunits (RI-Alpha, RI-Beta, RII-Alpha and RII-Beta), and three encode the C subunits (C-Alpha, C-Beta and C-Gamma). Although there are major differences in the tissue distribution, biochemical and physical properties of the R subunit isoforms, differences between the various isoforms of the C subunit are more subtle. PKA is classified as a Type I or Type II enzyme depending upon the associated R subunit (i.e., RI or RII). Type I PKA is predominantly located in the cytoplasm, while Type II associates with cellular structures and organelles. Type II PKA is not a “free floating” enzyme but is anchored to specific locations within the cell by specific proteins called AKAPs (A Kinase-Anchoring Proteins). The AKAPs keep PKA localized and limit the targets that can be phosphorylated, preventing the indiscriminate phosphorylation expected from free PKA in the cytoplasm. Most AKAPs described thus far bind the RII subunits with nanomolar affinities while binding RI subunits in the micromolar range. RII-binding AKAPs range in size from 15–300kDa and are capable of binding other kinases as well as phosphatases. Anchored PKA modulates the activity of various cellular proteins, including AMPA/Kainate channels, Glutamate receptor-gated ion channels, L-type Ca2+ channels in skeletal muscle, hormone-mediated Insulin secretion in clonal beta cells, Vasopressin-mediated translocation of Aquaporin-2 into the cell membrane of renal principal cells, motility of mammalian sperm and the sperm Acrosome reaction (Ref.1 & 2).
Regulation of PKA in the cell is related primarily to modulation of its phosphotransferase activity. The holoenzyme contains two C subunits bound to homo- or heterodimers of either RI or RII subunits. The C subunits do not interact with one another. The R subunits each have an N-terminal dimerization domain and two cAMP binding sites. Activation proceeds by the cooperative binding of two molecules of cAMP to each R subunit, which causes the dissociation and subsequent activation of each C subunit from the R subunit dimer. cAMP is a cyclic nucleotide that serves as an intracellular and, in some cases, extracellular “second messenger” mediating the action of many peptide or amine hormones. The level of intracellular cAMP is regulated by the balance between the activity of two types of enzyme: AC (Adenylyl Cyclase) and the cyclic nucleotide PDE (Phosphodiesterase). Several receptors are responsible for the activation of cAMP-PKA pathway, GPCRs (G-Protein Coupled Receptors) being the most common receptors. GPCRs include Alpha and Beta-ADRs (Adrenergic Receptors), CRHR (Corticotropin Releasing Hormone Receptor), GcgR (Glucagon Receptor), Smo (Smoothened) etc. All these receptors are responsible for cAMP accumulation in cells that cause different physiological outcomes and changes in cAMP levels effects the selective activation of PKA isoforms. The chief source of cAMP is from ATP (Adenosine Triphosphate). In mammals, the conversion of ATP to cAMP is mediated by members of the Class-III AC/ADCY (Adenylate Cyclase) family, which in humans comprises nine trans-membrane AC enzymes or tmACs and one soluble AC or sAC. tmACs are regulated by heterotrimeric G-proteins in response to the stimulation of various types of GPCRs and therefore play a key role in the cellular response to extracellular signals. sAC, in contrast, is insensitive to G-proteins. The extracellular stimuli like neurotransmitters, hormones, inflammatory stimuli, stress, epinephrine, norepinephrine, etc activate the G-proteins through GPCRs. The major G-proteins that regulate activation of ACs are the GN-AlphaS (GN-AlphaS Complex Locus), GN-AlphaQ (Guanine Nucleotide-Binding Protein-Alpha-Q) and GN-AlphaI (Guanine Nucleotide Binding Protein-Alpha Inhibiting Activity Polypeptide). Upon activation these subunits are separated from GN-Beta (Guanine Nucleotide-Binding Protein-Beta) and GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) subunits and are converted to their GTP bound states that exhibit distinctive regulatory features on the nine tmACs in order to regulate intracellular cAMP levels (Ref.1 & 4). Other ligands like Gcg (Glucagon) and Ucns (Urocortins) also regulate activity of ACs via G-protein activation through their respective receptors like GcgR and CRHR. GN-AlphaS and GN-AlphaQ activate ACs to increase intracellular cAMP levels, where as, GN-AlphaI decrease intracellular cAMP levels by inhibiting ACs. GN-Beta and GN-Gamma subunits act synergistically with GN-AlphaS and GN-AlphaQ only to activate ACII, IV and VII. However the Beta and Gamma-subunits along with GN-AlphaI inhibit the activity of ACI, V and VI. ACI, III, V, VI and IX isoforms play a vital role in spinal pain transmission and are up-regulated by chronic Opoids, for which they are often inhibited by Ca2+ and other proteins acting downstream to cAMP signaling. G-proteins indirectly influence cAMP signaling by activating PI3K and PLC (Phospholipase-C). PLC cleaves PIP2 (Phosphatidylinositol 4,5-bisphosphate) to generate DAG (Diacylglycerol) and IP3 (Inositol 1,4,5-trisphosphate). DAG in turn activates PKC (Protein Kinase-C). IP3 modulates proteins upstream to cAMP signaling with the release of Ca2+ through IP3R (IP3 Receptor). Ca2+ release activates Caln (Calcineurin) which facilitates NFAT (Nuclear Factor of Activated T-Cells) translocation to the nucleus, a process that is quite essential for axonal growth. Caln also inhibits ACIX thus modulating cAMP Signaling (Ref.3, 4 & 5).
cAMP, once formed, serves to modulate inotropy, chronotropy and lusitropy by inducing PKA phosphorylation of contractile proteins, ion channels, enzymes of intermediary metabolism and other regulatory proteins. Though cAMP is the major activator of PKA, PKA can also be formed independent of cAMP. Vasoactive peptides Endothelin-1 and AngiotensinII activate PKA by inducing phosphorylation and degradation of the inhibitor of IKappaB, subsequently releasing PKAc from inhibition by IKappaB. GN-Alpha13, upon interaction with protein kinase AKAP110 (A-Anchoring Protein-110), induces release of the PKAc from the AKAP110-PKAr complex, resulting in the PKA activation. GN-Alpha13 activates MEKK1 (MAPK/ERK Kinase Kinase-1) and RhoA via two independent pathways, which induce phosphorylation and degradation of IKappaB-Alpha, presumably through activation of IKK (IKappaB Kinase), leading to release and activation of PKA catalytic subunit. PKAc can be regulated by mechanisms that are cAMP independent. The phosphorylation of the p65/RelA subunit of transcription factor NF-KappaB that is catalyzed by PKAc is independent of cAMP. NF-KappaB is maintained in an inactive state in the cytosol by association with the inhibitor protein IKappaB. The catalytic subunit of PKA is also inactivated by binding to IKappaB, forming an NF-KappaB–IKappaB–PKAc complex. Upon lipopolysaccharide stimulation, IKappaB Kinase phosphorylates IKappaB, thereby targeting it for proteasomal degradation. PKAc is released in an active form and phosphorylates p65/RelA subunit of NF-KappaB on Ser-276, resulting in increased transactivating activity of NF-KappaB independent of nuclear translocation and increased DNA binding. Rather, increased transactivation is due to enhanced binding to the transcriptional coactivators, CBP/p300. PKAc also lead to VASP (Vasodilator-Stimulated Phosphoprotein) phosphorylation. PKA also activates NFAT5, independent of cAMP. PKAc stimulation of NFAT5 transactivating activity could be similar to that found for NF-KappaB (Ref.6 & 7).
PKA, once activated can phosphorylate several other substrates. The ADR-Alpha/Beta stimulation of cAMP-PKA phosphorylates several proteins related to excitation-contraction coupling like activation of RyR (Ryanodine Receptor), Pln (Phospholamban), CRP (C-Reactive Protein) but inhibits TnnI (Troponin-I). PKA phosphorylates Pln that regulates the activity of SERCA2 (Sarcoplasmic Reticulum Ca2+-ATPase-2). It leads to increased reuptake of Ca2+, Cl- (Chloride Ions), K+ (Potassium Ions), Na+ (Sodium Ions) and this process is affected in failing hearts. Phosphorylation of TnnI desensitizes contractile proteins to Ca2+, thus providing a mechanism for relaxation. PKA also activates KDELR (KDEL (Lys-Asp-Glu-Leu) Endoplasmic Reticulum Protein Retention Receptor) to promote retrieval of proteins (protein retention) from golgi complex to endoplasmic reticulum thereby maintaining steady state of the cell. Increase in higher concentration of Ca2+ and PKA activation enhances eNOS (Endothelial NOS) enzyme activity by phosphorylation of Serine residue (Ser635) in order to stimulate eNOS signaling, which is essential to maintain cardiovascular homeostasis (Ref.13). cAMP activated PKA represses ERK (Extracellular Signal-Regulated Kinase) activation by the formation of an inactive Rap1 (Ras-Related Protein-1)/ Raf1 (v-Raf1 Murine Leukemia Viral Oncogene Homolog-1) that interferes with ERK activation through seizure of MEK1,2 (MAPK/ERK Kinase-1/2) activation by sequestering Raf1 activity. Therefore, the effect of cAMP on ERK differs depending on the balance of the Raf1, BRaf (v-Raf Murine Sarcoma Viral Oncogene Homolog-B1) and PKA isoforms occurring inside a cell (Ref.10). cAMP can activate Rap1, either by direct stimulation of GEFs (Guanine nucleotide Exchange Factors), or via PKA. Moreover, PKA may also be required for neurotrophin-mediated activation of Rap1. Long PDE4A (cAMP Specific Phosphodiesterase-4A) isoforms are also activated by PKA, that sequester cAMP activity by converting it back to AMP. PKA inhibit the interaction of 14-3-3 proteins with BAD (Bcl2-Antagonist of Cell Death) (through 14-3-3/BAD signaling) and NFAT to promote cell survival (Ref.8, 9 & 11).
Activated PKA phosphorylates endothelial MLCK (Myosin Light Polypeptide Kinase), thereby reducing its activity, leading to decreased basal MLC (Myosin Light Chain) phosphorylation. Elevation of intracellular cAMP levels and activation of PKA stimulate phosphorylation of the Actin-binding proteins Filamin and Adducin and focal adhesion proteins Paxillin and FAK (Focal Adhesion Kinase), as well as the disappearance of stress fibers and F-actin (Filamentous Actin) accumulation in the membrane ruffles. PKA-mediated modulation of Rho GTPase activity is another potentially important mechanism for regulation of Actin cytoskeletal organization. Elevation of intracellular cAMP and increased PKA activity attenuates RhoA activation via RhoA phosphorylation at Ser188, which decreases Rho association with Rho-Kinase. Rho Kinase regulates Myosin-II and cell contraction by catalyzing phosphorylation of the regulatory subunit of Myosin phosphatase, PPtase1 (Protein Phosphatase-1), by inhibiting its catalytic activity, which results in an indirect increase in RLC (Regulatory Light Chain of Myosin) phosphorylation. Inactivation of Rho Kinase also directly increases RLC phosphorylation. PKA activation also increases interaction of RhoA with Rho-GDI (Rho-GDP Dissociation Inhibitor) and translocation of RhoA from the membrane to the cytosol. Thus the overall effect of PKA on RhoA is the inhibition of RhoA activity and stabilization of cortical Actin cytoskeleton. PKA also controls phosphatase activity by phosphorylation of specific PPtase1 inhibitors, such as DARPP32 (Dopamine-and cAMP-Regulated Phosphoprotein). Neurotransmitters enhance DARPP32 interaction via GPCRs, which leads to suppression of PPtase1 activity, when DARPP32 is phosphorylated at Thr-34 (Threonine-34) position. PPtase1 checks the phosphorylation of CREB (cAMP Responsive element binding Protein), CREM (cAMP Response Element Modulator) and ATF1 (Activating Transcription Factor-1) so that they are able to interact with the co-activators like CBP (CREB-Binding Protein) and p300. PKA also activates CREB and controls the expression of critical genes such as BDNF (Brain-Derived Neurotrophic Factor). Similarly, phosphorylation of NF-KappaB (Nuclear Factor-KappaB) by PKA is necessary for transcriptional activation and interaction with CBP (Ref.10 & 11).
Serine 21 in GSK3Alpha (Glycogen Synthase Kinase-3-Alpha) and Serine 9 in GSK3Beta (Glycogen Synthase Kinase-3-Beta) are also physiological substrates of PKA. PKA physically associates with, phosphorylates, and inactivates both isoforms of GSK3, thus prevents Oncogenesis and neurodegeneration. HSL (Hormone-Sensitive Lipase), an important enzyme of lipolysis, is also phosphorylated by PKA. PKA-phosphorylated HSL rapidly translocates and adheres to the surface of lipid droplets. It is this translocation and not HSL activation that accounts for the strong lipolytic enhancement following PKA activation. PKA interferes at different levels with other signaling pathways. Inactivation of PTP (Protein Tyrosine Phosphatase) results in dissociation from and consequent activation of ERKs. Inactivation of PCTK1 (PCTAIRE Protein Kinase-1) and APC (Anaphase-Promoting Complex) helps to maintain control cell proliferation and anaphase initiation and late mitotic events, respectively, thereby checking the degradation cell cycle regulators. Gcg binds to GcgR/GN-Alpha proteins on the surface of liver cells and triggers an increase in cAMP production leading to an increased rate of Glycogenolysis by activating PHK (Phosphorylase Kinase) via the PKA-mediated cascade. PHK further activate PYG (Glycogen Phosphorylase), which converts Glycogen to Glucose-1-Phosphate. Phosphoglucomutase then transfers phosphate to C-6 of Glucose-1-Phosphate generating Glucose-1,6-phosphate as an intermediate. The phosphate on C-1 is then transferred to the enzyme regenerating it and Glucose-6-Phospahte is the released product that enters Glycolysis. This is the same response hepatocytes have to Epinephrine release through the ADR-Alpha/Beta. PKA further inhibits GYS (Glycogen Synthase) leading to seizure of energy consuming process like Glycogen Synthesis. PKA phosphorylates GRK1 (G-Protein-Dependent Receptor Kinase-1) at Ser(21) and GRK7 (G-Protein-Dependent Receptor Kinase-7) at Ser(23) and Ser(36). Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate Rhodopsin (Ref.10, 12 & 13).
KA modulates the activity of transcription factors, such as nuclear receptors and HMG (High Mobility Group)-containing proteins, influencing their dimerization or DNA-binding properties. PKA also regulates Gli3 (Gli-Kruppel Family Member-3) under the influence of Hedgehog signaling. Function of Gli3 is similar to that of Drosophila gene CI (Cubitus Interruptus) activity. In this case phosphorylation stimulates a specific cleavage of Gli3 which transforms the protein from an activator to a repressor. However, proteins like PKIs (Protein Kinase Inhibitors) and Mep1B (Meprin-A-Beta) down regulate PKA activity to prevent aberrant gene expression. In mammalian cells, including human, PKA regulate a huge number of processes, including growth, development, memory, metabolism, and gene expression. Failure to keep PKA under control can have disastrous consequences, including diseases such as cancer. Drugs based on inhibiting PKA activity are under development for treating disease, so understanding how cAMP accomplishes this task is of interest to life scientists (Ref.14 & 15).