| cAMP (Cyclic Adenosine 3',5'-monophosphate) is the first
identified second messenger, which has a fundamental role in the
cellular response to many extracellular stimuli. The cAMP signaling
pathway controls a diverse range of cellular processes. Indeed, not only
did cAMP provide the paradigm for the second messenger concept, but also
provided the paradigm for signaling compartmentalization. The different
receptors, chiefly the GPCRs (G-Protein Coupled Receptors), Alpha and
Beta-ADRs (Adrenergic Receptors), Growth Factor 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 (cAMP dependent Protein
Kinase-A) 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 (Adenylyl Cyclase)/ADCY (Adenylate Cyclase)
family (E.C 4.6.1.1), which in humans comprises nine trans-membrane AC
enzymes or tmACs and one soluble AC or sAC (Ref.1 & 2). The sAC
predominantly occurs in mature spermatozoa. 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.
Instead, sAC is directly activated by Ca2+ (Calcium Ions) and the
metabolite HCO3- (Bicarbonate Ions), rendering the enzyme an
intracellular metabolic sensor. Together, tmACs and sAC regulate a
diverse set of essential biological processes, such as differentiation
and gene transcription, and this makes cAMP signaling, an important
mediator of intra- and extracellular signals in organisms from
prokaryotes to higher eukaryotes (Ref.2 & 3).
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 (Ref.1 &
4). 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. GN-AlphaI activity is counteracted by RGS (Regulator of
G-Protein Signaling) proteins (Ref.5, 6 & 7). However, Beta-Arrestins
desensitize many GPCRs and ADRs and this blocks interaction between
receptors and their cellular effectors, thereby inhibiting GPCR
activity. Beta-Arrestins contol intracellular cAMP levels by switching
of signaling from GN-AlphaS to GN-AlphaI by recruiting PDE4D (cAMP
Specific Phosphodiesterase-4D) isoforms (Ref.8). Growth factors and PI3K
(Phosphatidylinositde-3 Kinase) crosstalk with cAMP signaling by
activating Akt (v-Akt Murine Thymoma Viral Oncogene Homolog), which
further activates PDE (Phosphodiesterase) to facilitate the conversion
of cAMP to AMP through Akt Signaling. This modulates cardiac
contractility and release of metabolic energy. 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)
facilitating activation of Src (v-Src Avian Sacroma (Schmidt-Ruppin
A-2)Viral Oncogene) along with PYK2 (Proline-Rich Tyrosine Kinase-2).
Activation of Src by PI3K and growth factors enhance activation of Raf1
(v-Raf1 Murine Leukemia Viral Oncogene Homolog-1) through Ras. Raf1
facilitate MEK1 (MAPK/ERK Kinase-1) and MEK2 activation, which in turn
activates ERKs (Extracellular Signal-Regulated Kinases) and this
ultimately leads to induction of transcription regulator Elk1 (ETS-domain
protein Elk1) mediated gene expression. PKC modulate cAMP signaling by
activation of Raf1, PYK2 and ACs like ACI, II, III, V and VII, but
inhibits ACVI (Ref.9).
Once active, the tmACs and sAC produces the second messenger cAMP in
response to a wide range of signal transduction pathways. Three main
targets of cAMP are PKA, the GTP-exchange protein, EPACs (Exchange
Protein Activated by cAMP) and the CNG (Cyclic-Nucleotide Gated Ion
Channel). CNG activation by cAMP provides passage to Ca2+ influx. cAMP
activate Rap1A (Ras-Related Protein-1A) and Rap1B (Ras-Related Protein
Rap1B) through the PKA-independent and EPAC (Exchange Protein Activated
by cAMP)-dependent pathway. cAMP activates cAMP-GEFI (cAMP-Regulated
Guanine Nucleotide Exchange Factor-I)/EPAC1 and cAMP-GEFII (cAMP-Regulated
Guanine Nucleotide Exchange Factor-II)/EPAC2 that in turn activate Rap1A
and Rap1B, respectively. Rap1A and Rap1B then forms an active complex
with BRaf (v-Raf Murine Sarcoma Viral Oncogene Homolog-B1) for MEK1/2
activation finally resulting in Elk1 activation. Rap1A and Rap1B further
stimulate Rap1 and Rap2 pathways that are vital for cell survival
(Ref.10 & 11). Apart from CNG, PKC, and EPACs, other direct targets
of cAMP includes, PDE, mTOR (Mammalian Target of Rapamycin),
p70S6K/RPS6KB1 (Ribosomal Protein-S6 kinase-70kDa-Polypeptide-1), PLA2
(Phospholipase-A2). cAMP-activated mTOR and p70S6K promote cell growth
via the mTOR and p70S6K signaling route, whereas, PLA2 facilitates
release of stored energy by enhancing the Fatty acid metabolism
processes. The Urocortin-cAMP mediated induction of PKC and p38 results
in Apoptosis and Cytokine production (like that of IL-6
(Interleukin-6)), downstream to the Urocortin-cAMP pathway. Although
cAMP directly regulates the activities of some molecules, PKA appears to
be the major 'read-out' for cAMP and is the predominant cellular
effector of cAMP (Ref.12). PKA is tethered to specific cellular
locations by a growing class of proteins called AKAPs (A-Kinase Anchor
Proteins). Targeting of PKA isozymes by AKAPs is important for an
increasing number of physiological processes such as cAMP regulation of
ion channels in the nervous system, regulation of the cell cycle which
involves microtubule dynamics, chromatin condensation and decondensation,
nuclear envelope disassembly and reassembly, Steroidogenesis,
reproductive function, immune responses and numerous intracellular
transport mechanisms (Ref.13).
In the following sections, the role of localized pools of PKA in the
context of some selected physiological processes where regulation by
cAMP plays a major role has been analyzed. 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 inhibts 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. Ca2+ uptake activates Caln (Calcineurin),
which further facilitates NFAT (Nuclear Factor of Activated T-Cells)
translocation to the nucleus, a process that is quite essential for
axonal growth. cAMP plays a vital role in regulation of cardiovascular
function by controlling the process of myocardial contraction. 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). In mammals, Ca2+ and HCO3-
play a critical role in the regulation of sperm function, most likely by
regulation of cAMP levels. sAC is active in human spermatozoa and is a
sensor for both HCO3- and Ca2+. Ca2+ release by CaCn and CNG, activate
Calm (Calmodulin) and CamKs (Calcium/Calmodulin-Dependent Protein
Kinases). Calm further activate Caln, CamK2 (Calcium/Calmodulin-Dependent
Protein Kinase-II) and CamK4. These in turn modulate cAMP production by
regulating the activity of ACs and PDEs. The CamKs along with Caln
inhibit PDE and ACIX, whereas CamK2 and CamK4 inhibit the function of
ACIX and ACI, respectively (Ref.2). ACIX is also inhibited by PKC thus
controlling cAMP signaling in the hippocampal neurons. cAMP activated
PKA represses ERK activation by the formation of an inactive Rap1/Raf1
that interferes with ERK activation through seizure of MEK1,2 activation
by sequestering Raf1 activity. 14-3-3 like the PKA also aids the process
of Raf1 inactivation. Therefore, the effect of cAMP on ERK differs
depending on the balance of the Raf1, BRaf and PKA isoforms occurring
inside a cell (Ref.10). Long PDE4A (cAMP Specific Phosphodiesterase-4A)
isoforms are activated by cAMP, PKA and Akt kinases. The PDE sequester
cAMP activity by converting it back to AMP. This negative-feedback loop
terminates the cAMP signal locally. cAMP further represses the activity
PDE1 (Phosphodiesterase-1) to enhance the duration and intensity of cAMP
signaling (Ref.14).
cAMP follows a distinct route and activates a single PKA-AKAP complex
close to the substrate to mediate a distinct biological effect.
Accordingly, each substrate appears to have its own, private anchored
pool of PKA and its own local gradient of cAMP. PKA inhibit the
interaction of 14-3-3 proteins with BAD (through 14-3-3/BAD signaling)
and NFAT to promote cell survival. PKA inhibits Adducin action by
limiting its role during assembly of Spectrin-Actin network in
erythrocytes, thereby reducing the chances of Erythroleukemia. It
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. Increased cAMP levels promote survival of neuronal
cells by inactivating GSK3Alpha (Glycogen Synthase Kinase-3-Alpha) and
GSK3Beta (Glycogen Synthase Kinase-3-Beta) via a PKA dependent mechanism
and thus prevents Oncogenesis and neurodegeneration (Ref.13 & 15).
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. PKA activation by cAMP enhances release of stored
energy in cells by phosphorylation of HSL (Hormone-Sensitive Lipase) in
white adipose tissue, which leads to the hydrolysis of triglycerides
(vital intermediates of Tricaylglycerol Metabolism). Hydrolysis of
triglycerides by HSL generates free fatty acids, the major gateway for
the release of stored energy, and this process is termed as Lipolysis.
Gcg binds to 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 (Ref.13 & 16).
Upon stimulation from hormones, cAMP increases phosphorylation of
RhoA that inactivates 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. Such increased
intracellular cAMP and PKA activation on RLC phosphorylation decreases
Thrombin-induced isometric tension development in endothelial cells and
this decrease the development of Edema, a hallmark of acute and chronic
inflammation (Ref.17). Interestingly, PKA 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. Phosphorylation is a crucial event in transcriptional
activation by CREB (cAMP Response Element-Binding Protein), CREM (cAMP
Response Element Modulator) and ATF1 (Activating Transcription
Factor-1), because it allows interaction with the transcriptional
co-activators CBP (CREB-Binding Protein) and p300. CREM gene encodes
many different isoforms, some of which have repressive functions.
Particularly the repressor ICER (Inducible cAMP Early Repressor),
participates in the downregulation of cAMP-induced transcription by
competing with the binding of CREB and CREM activators to their DNA
binding sites. PPtase1 checks the phosphorylation events in order to
inactivate the formation of repressor isoforms like ICER so that CREB,
CREM and ATF1 are able to interact with the co-activators like CBP and
p300. Hence, under physiological conditions, ICER induction is a
transient phenomenon that allows cAMP signaling to return to the basal
state. In contrast, prolonged or inappropriate induction of ICER elicits
pathological consequences (Ref.18). Similarly, phosphorylation of
NK-KappaB (Nuclear Factor-KappaB) by PKA is necessary for
transcriptional activation and interaction with CBP. 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. A peculiar example is the
mechanism by which PKA regulates Gli3 (Gli-Kruppel Family Member-3)
under the influence from 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 (Ref.13
& 16).
Normally, the level of intracellular cAMP is regulated by the balance
between the activities of two types of enzymes; ACs and the cyclic
nucleotide PDEs chiefly in response to hormones and neurotransmitters.
The cAMP signaling is involved in controlling exocytotic events in
polarized epithelial cells with implication for Diabetes Insipidus,
Hypertension, Gastric ulcers, Thyroid disease, Diabetes Mellitus, and
Asthma. Heterologous sensitization of cAMP signaling contributes to
fundamental physiological processes such as the timing of circadian
rhythms, sexual behavior, and neurotransmitter crosstalk, and also to
neurological disorders such as substance abuse and drug-induced
Dyskinesias (Ref.19). This provide insight into the mechanisms of
addiction and many other central nervous system disorders reflective of
altered neuronal cell functioning. Besides it also represent very likely
roles for ACs and open up avenues for therapeutics targeting ACs that
may prove useful in the development of male contraceptives (Ref.1 &
20). |
