CYCLIC NUCLEOTIDES: CYCLIC AMP AND PROTEIN KINASE A
Cyclic AMP (cAMP)
- Synthesized from ATP by plasma membrane-bound adenylate cyclase.
- Degraded to 5-AMP by cycle AMP-specific phosphodiesterase.
- To function as intracellular messenger, its concentration (normally <10 M,
which is low) must be able to change up or down rapidly in response to
extracellular signals Ex. on hormonal stimulation, cAMP levels can
change at least 5-fold in few seconds in a step-like fashion.
o Constantly competing forces: step-like up, curve down.
- The primary physiological function of cAMP is to serve as an intracellular
second messenger to bind to and activate protein kinase A (PKA).
- Another physiological role for cAMP identified more recently is to act as a
ligand for a specific class of odorant cation channel in olfactory neurons
(intracellular in this case, so responds to signals/changes in environment
inside the cell).
Figure: Not a homogenous process (localizes in one part of the cell).
- Adenylate cyclase removes two phosphates to create cAMP.
- Degradation to 5-AMP by cyclic AMP-specific phosphodiesterase.
Adenylate Cyclase (first enzyme in cascade to convert ATP into cAMP)
- Consists of two alternating hydrophobic and hydrophilic domains.
- Hydrophobic domains each contain 6 membrane-spanning domains.
- Hydrophilic regions have two catalytic domains.
- Multiple isoforms of adenylate cyclase stimulated by Gs and inhibited by Gi.
- At least one isoform complexes with Ca bound to calmodulin leading to modulation of enzyme activity.
Figure: Membrane-bound enzyme mostly at plasma membrane.
Two alternating hydrophobic and hydrophilic domains.
Two catalytic domains used per GPCR each with 6 membrane
Cyclic AMP-dependent Protein Kinase A (PKA)
a) a) Phosphorylation of Cellular Proteins
- Inactive PKA consists of a heterotetrameric
complex with 2 catalytic and 2 regulatory
subunits that have high affinity for each
- Increase in cAMP in response to activation of
adenylate cyclasePKA floats in cytoplasm.
- 2 cAMPs bind to each regulatory - Cyclic AMP binds allosterically.
subunit leading to conformational - Conformation lowers the affinity of subunits for each other.
rearrangement and dissociation
from the complex. - Catalytic subunit transfers phosphates to proteins.
- Need 4 cAMP molecules for full activity. Activation of cAMP-dependent PKA
- Binding of cAMP to regulatory subunits induces a conformational change causing subunits to dissociate
leading to activation of catalytic subunits.
- Each regulatory subunit has 2 cAMP binding sites.
- Release of catalytic subunits requires binding of more than 2 cAMP molecules to the tetramer.
- Allosteric mechanism enhances response of PKA to change in cAMP concentration.
o 2 regulatory subunits with 2 cAMP and conformational changes make it easier for other cAMP to bind.
- Amplification: smaller amount of starting material to more ending material (2 active catalytic subunits).
- Released catalytic subunits are activated to phosphorylate specific substrate proteins.
- When catalytic subunits are freed and active they can:
o Phosphorylate cytoplasmic proteins
o Migrate into the nucleus to phosphorylate transcription factors
o Regulatory subunits remain in the cytoplasm
- Less likely to bind cAMP if wait too long (step-like up, curve down constantly)temporal and special aspects.
- Role of cAMP and PKA in regulation of
biological activity of cytoplasmic proteins.
- PKA catalyzes transfer of terminal
phosphate group from ATP to specific Ser
and Thr residues of substrate proteins.
- Usually excitatory, but can be inhibitory.
- Remain active in nucleus until degraded or
move back to cytoplasm (lasts longer in
nucleus than in cytoplasm).
Inactivation of cAMP-dependent PKA
1. cAMP concentration in cell returns to normal then get reassociation of regulatory and catalytic subunits.
2. Catalytic subunits can be inhibited by endogenous cellular proteins called protein kinase inhibitors (PKIs)
o PKIs bind irreversibly to catalytic subunits thereby inhibiting their catalytic functionscell can
make more catalytic subunits later to compete because bound irreversibly.
o Precise physiological roles note entirely knownbut cell can generate lots if needed.
o PKIs appear to compete with regulatory subunits for binding to and inactivaton of catalytic subunits.
b) Activation of Gene Transcription
- Major discover in the field of cell signaling: identification of DNA-binding proteins whose transcriptional
functions are activated by phosphorylation by PKA.
- These bind specific DNA elements located in many gene promoters.
- These DNA binding elements are called CRE (cAMP response element) and the TF is called CREB (cAMP
response element binding protein)others do exist.
o Member of large superfamily of DNA-binding proteins (bZIP).
o DNA-binding domains consist of basic region (b) that recognizes and binds to DNA, and Leu zipper
(ZIP) with long repeats of Leu residues responsible for dimerization (always work as dimers!! Either
homo or hetero).
o Transcriptional activity of CREB is regulated by cAMP-dependent signaling pathways, and represents
final communication link in regulation of gene expression by activation of this pathway. Figure: CREB is substrate for PKA and in phosphorylated state
it can bind to CRE and activate transcription.
Catalytic subunit can translocate into the nucleus where it is
protected from rebinding to regulatory subunits (that is why
duration is longer in nucleus).
- Role of PKA and CREB in regulation of proinsulin gene
transcription in pancreatic beta cells.
- Regulatory subunits anchored on kinase anchor protein
(KAP), which stabilize subunits in certain places in
- Activated adenylyl cyclase (net activity determines its activity).
- Cholera Toxin Acts on glucagon receptor to activate adenylyl cyclase activity.
- Pertusses Toxin Opposite effect, inhibiting adenylyl cyclase.
- GLP-I (glucagon-like peptide) and Galanin work in +/- fashion to determine how much insulin to produce
(bind to own receptorshave own G-proteins).
- Experimental agents used to study these processes (see if something is done by cAMP):
o Forskolin can bind directly to AC (adenylyl cyclase) and activate it, causing increase in cAMP (tells us if
its involved directly).
o Analogues (mimics) of cAMP Diffuse into cell (caffeine has same effect).
o cAMP mimic that can diffuse into nucleus (8-bromocyclic AMP) not degraded by phosphodiesterase
(PDE), so activation would be sustained.
o Xanthine analog (caffeine for example) inhibits PDE which would again sustain cAMP effect.
o IBMX Mimics PDE preventing cAMP AMP prolonging PKA function.
Representative Cellular Actions of cAMP-Dependent PKA:
a) Glycogen Phosphorylase and Glucose Metabolism
- Circulating adrenaline causes muscle cells to break glycogen to glucose-1-phosphate and stop making glucose.
- Glucose is oxidized by glycolysis to provide ATP for sustained muscle contraction.
- Mechanisms: Adrenaline causes -adrenergic receptor-induced increase in cellular cAMP causing PK-A to
phosphorylate 2 other specific enzymes:
1. Phosphorylase Kinase (Phosphorylates glycogen phosphorylase which is activated to release glucose
residues from glycogen molecules).
2. Glycogen Synthase (Performs final step in glycogen synthesis from glucose, and is inhibited by
phosphorylation to shut off glycogen synthesise.g. of phosphorylation not activating). b) Phosphatases Rapidly Reverse Effects of PKA
- Dephosphorylation of phosphoryalated Ser and
Thr residues is catalyzed by a group of 4 Ser/Thr
- Protein Phosphatase-1 (PP-1) plays an