PKA (Protein Kinase-A) is an enzyme that regulates processes as diverse as growth, development, memory, and metabolism. In its inactivated state, PKA exists as a tetrameric complex of two Catalytic subunits (PKA-C) and a Regulatory (PKA-R) subunit dimer. To date, four regulatory subunits have been identified (RI-Alpha, RI-Beta, RII-Alpha and RII-Beta), which are differentially distributed in mammalian tissues. RI-Alpha and RII-Alpha are expressed ubiquitously, RI-Beta is expressed predominantly in the brain and RII-Beta is expressed primarily in brain, adrenal and adipose tissues. These R subunits define Types-I and II PKA, with both types of holoenzyme having three potential C subunits (Alpha, Beta and Gamma). The C-Alpha and Beta subunits share 93% homology and have broad tissue specificity, with C-Alpha being the predominant species. The C-Gamma subunit, however, has been readily identified only in the primate testis. 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). When both binding sites on the R subunits are occupied by cAMP (Cyclic Adenosine 3,5-Monophosphate), the R subunits undergo a conformational change, which lowers their affinity for the C subunits. This results in the dissociation of the holoenzyme complex and renders the enzyme active. The catalytic subunits are then free to phosphorylate specific target proteins. Thus, PKA is activated by cAMP to transfer phosphates from ATP (Adenosine Triphosphate) to protein substrates (Ref.1 & 2).
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). The different receptors, chiefly the GPCRs (G-Protein Coupled Receptors), Alpha and Beta-ADRs (Adrenergic Receptors), CRHR (Corticotropin Releasing Hormone Receptor), GcgR (Glucagon Receptor), DCC (Deleted in Colorectal Carcinoma), etc are responsible for cAMP accumulation in cells that cause different physiological outcomes, and changes in cAMP levels effects the selective activation of PKA isoforms. 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. tmAC 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. Instead, sAC is directly activated by Ca2+ (Calcium Ions) and the metabolite HCO3- (Bicarbonate Ions), rendering the enzyme an intracellular metabolic sensor. The extracellular stimuli like neurotransmitters, hormones, inflammatory stimuli, stress, epinephrine, norepinephrine, etc activate the G-Proteins through receptors like GPCRs and ADR-Alpha/Beta. 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. Other ligands like Gcg (Glucagon), Ucns (Urocortins) and Ntn1 (Netrin-1), etc either directly regulate activity of ACs or via G-Protein activation through their respective receptors like GcgR, CRHR and DCC. 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 (Ref.2 & 3). 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+ is also released by CaCn and CNG. 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. Ca2+ release also activates Calm (Calmodulin), CamKKs and CamKs (CamK4 and CamK2). CamK4 and CamK2 phosphorylate CBP (CREB Binding Protein) and Histone Deacetylases, HDAC4, HDAC5 and HDAC7, which mediates some nuclear Ca2+ signals. CamKs also take part in cAMP modulation by inhibiting ACI. CamKs also inhibit PDE that sequester cAMP activity by converting it back to AMP (Adenosine Monophosphate). In T-Cells, the engagement of both the TCR (T-Cell Receptor)-CD3 complex and the CD28 co-stimulatory molecule also induces cAMP (Ref. 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 Angiotensin-II 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 PKAc. 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. PKAc is released in an active form and phosphorylates p65/RelA subunit of NF-KappaB on Ser276, 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 (Ref.6). PKAc also lead to VASP (Vasodilator-Stimulated Phosphoprotein) phosphorylation. High levels of AMP under stress conditions like hypoxia, ischemia and heat shock can also directly activate PKA. Certain Growth factors like TGF-Beta (Transforming Growth Factor-Beta) also activates PKA independent of cAMP. TGF-Beta directly binds to TGF-BetaR2 (Transforming Growth Factor-Beta Receptor-Type II), which leads to the phosphorylation of TGF-BetaR1 (Transforming Growth Factor-Beta Receptor-Type-I). This phosphorylation activates the RI protein kinase, which then phosphorylates SMAD3 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-3). Phosphorylated SMAD3 binds to SMAD4 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-4), and this complex binds to the regulatory subunits of PKA, leading to the release of catalytic subunits and resulting in the activation of downstream target genes (Ref.7 & 8).
PKA, once activated can phosphorylate several substrates. The ADR-Alpha/Beta stimulation of cAMP-PKA phosphorylates several proteins related to excitation-contraction coupling like activation of L-Type CaCn (Calcium Channels), KCn (Potassium Channel), SCn (Sodium Channels), ClCn(Chloride Channels), 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 Ser635 in order to stimulate eNOS signaling, which is essential to maintain cardiovascular homeostasis. 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.9, 10 & 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 GTPases 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 Thr34 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. Other substrates of PKA include Histone H1, Histone H2B and CREB (Ref.12, 13 & 16).
Ser21 in GSK3Alpha (Glycogen Synthase Kinase-3-Alpha) and Ser9 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 preventing 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 cell proliferation and anaphase initiation and late mitotic events, respectively, thereby checking the degradation cell cycle regulators (Ref.14). PKA phosphorylates and inactivates GYS (Glycogen Synthase), which prevents the futile cycling of Glucose-1 Phosphate back into Glycogen via UDP-glucose. PKA phosphorylates GRK1 (G-Protein-Dependent Receptor Kinase-1) at Ser21 and GRK7 (G-Protein-Dependent Receptor Kinase-7) at Ser23 and Ser36. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate Rhodopsin. PKA also phosphorylates Beta-Catenin. Phosphorylation of Beta-Catenin by PKA inhibited ubiquitination of Beta-Catenin in intact cells and in vitro. Catalytic subunit of PKA also interacts with and phosphorylates p75(NTR). Phosphorylated p75(NTR)may translocate to lipid rafts, where a number of downstream signaling molecules such as RhoA and ceramide accumulate, thus facilitating the efficiency of the signal transduction (Ref.15, 16 & 17).
PKA 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.2 & 18).