Signaling Systems Using Cyclic Nucleotides as Mediators: Focus on the Regulation of Gene Transcripti

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Signaling Systems Using Cyclic Nucleotides as
Mediators Focus on the Regulation of Gene
Transcription by Glucagon

• David L. Morris

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Cyclic Nucleotides the Nobel Prize

• Hormonal action through cAMP- Sutherland (1971)
• Reversible protein phosphorylation- Fisher and
  Krebs (1992)
• G proteins cyclic nucleotide synthesis- Gilman
  and Rodbell (1994)
• Nitric oxide cGMP- Furchgott, Ignarro, and
  Murad (1998)
• Cyclic nucleotides the nervous system-
  Carlsson, Greengard, and Kandel (2000)
• Nobel e-museum http//www.Nobel.Se/index.Com

Beavo, J. and L. Brunton Nat Rev Mol Cell Biol
2002 Sep3(9)710-8
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cAMP/ cGMP mediate diverse physiological
processes

• Musculature
• Striated contraction
• Smooth muscle relaxation
• Cardiac contractility
• Energy/ electrolyte homeostasis
• Gluconeogenesis
• Glycolysis
• Lipogenesis
• Glomerular filtration
• Reproduction
• Testicular/ ovarian function
• Sperm mobility/ chemotaxis
• Acrosome reaction

• Nervous system
• Neurotransmission
• Learning and memory
• Phototransduction
• Olfaction
• Circadian rhythm
• Plasticity
• Cellular processes
• Proliferation
• Differentiation
• Ion transport
• Apoptosis

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Structures of Cyclic AMP/GMP
Beavo, J. and L. Brunton Nat Rev Mol Cell Biol
2002 Sep3(9)710-8
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Cyclic nucleotide synthesis/ degradation

• Cyclases convert ATP/GTP to cAMP/cGMP
• Adenylyl cyclases ATP? cAMP PPi
• 9 classes (transmembrane)
• Guanylyl cyclases GTP ? cGMP PPi
• Receptors for extracellular ligands
• Classified by number of transmembrane segments
  (zero, one, or multiple)
• Phosphodiesterases
• cleave phosphodiester bond (5 AMP, 5 GMP)

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Nucleotide interaction in cyclase active sites

• Lysine (K938) and Aspartate (D1018) in AC
  hydrogen bond to N-1 and N-6 of ATP
• Cysteine (C541) and Glutamate (E473) in GC
  contact O-6 and N-2 of GTP
• These residues are highly conserved and invariant
  in ACs and CGs

Wedel and Garbers. 2001 Annu. Rev. Physiol.
63215-233
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Receptor/ ligand activation of cyclases

• cAMP
• 7TM receptors
• G-coupled protein receptors activate/ inhibit AC
  by G?-GTP subunit interaction
• cGMP
• Nitric oxide binds soluble GC, activation
• Atrial and B-type natriuretic peptides bind
  natriuretic peptide receptors (NPR), activation
• NPRs have intrinsic GC activity (C-terminus)
• Enterotoxins bind GC in intestinal tract

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Effectors of cAMP/ cGMP action

• Protein kinases
• cAMP-dependent (PKA)
• cGMP-dependent (PKG)
• Multiple substrates
• Cyclic nucleotide binding proteins
• Cyclic nucleotide-gated channels
• Guanine-nucleotide exchange factors (GEFs)

• Phosphodiesterases (PDEs)
• cGMP () PDE2
• Modulate cyclic nucleotide concentrations
• Transcriptional regulation
• cAMP-response element binding protein (CREB)
• cAMP-response element (CRE) in DNA promoters

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Cellular Targets of cAMP/ cGMP
cAMP cGMP
Beavo, J. and L. Brunton Nat Rev Mol Cell Biol
2002 Sep3(9)710-8
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GCPRs and cAMP

• Touhara, K. Microsc Res Tech 2002 Aug
  158(3)135-41

Beavo, J. and L. Brunton Nat Rev Mol Cell Biol
2002 Sep3(9)710-8
Zaccolo et al. Curr Opin Cell Biol. 2002
Apr14(2)160-6. Review
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Enterotoxin activates cGMP/ cross-talk with cAMP
Lucas, K et al. 2000 Pharmacol Rev. 52375-413
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cAMP Dependent Protein Kinase (PKA)

• Regulation of cytosolic phosphoenolpyruvate
  carboxykinase-1 (PEPCK-C) gene expression by
  glucagon

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Glucagon

• Produced by pancreatic islets in response to
  hypoglycemia
• Counteracts insulin
• Promotes hepatic mobilization of glucose
• Regulates rate of glycolysis and gluconeogenesis
  by
• Phosphorylation of key enzymes
• Transcriptional regulation of key metabolic
  enzymes
• PEPCK-C regulation

Jiang and Zhang. Am J Physiol Endocrinol Metab
284E671-678, 2003
Activated cAMP dependent protein kinase A is
the primary mediator in both processes
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Jiang and Zhang. Am J Physiol Endocrinol Metab
284E671-678, 2003
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PKA

• Ser/ Thr Kinase
• Holoenzyme
• Tetramer
• 2 Regulatory subunits
• RI?,RI?,RII?,RII?
• Blocks the active site (C subunit)
• 2 Catalytic subunits
• C?, C?, C?
• Regulation by cAMP
• Binding of cAMP to R subunit reduces affinity of
  proteinprotein interaction

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RI? binds 2 molecules cAMP
cAMP
Su, Y. et al. Science 1995 Aug 11269(5225)807-13
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Catalytic subunit
ATP
Taylor, S. et al. Pharmacol Ther 1999
May-Jun82(2-3)133-41
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PKA subcellular location

• Phosphorylation of diverse substrates by PKA is
  dependent on subcellular location
• Type I holoenzyme (RI? and RI? dimer)-
  cytoplasmic
• Type II (RII? and RII? dimer)- associated with
  cellular structures and organelles
• Localization of Type II PKA is mediated by
  nonenymatic scaffold proteins, A-kinase anchoring
  proteins (AKAPs)
• Compartmentalization of PKA ensures coordinate
  regulation by cAMP gradients as generated by AC

Reviewed by Alto, E. et al. Diabetes 2002 Dec 51
Suppl 3S385-8
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AKAPs dictate cellular location of PKA in the
heart
Kapiloff, Michael S. Mol Pharmacol 2002 62
193-199
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Dissociation of PKA holoenzyme

• cAMP activation of PKA can be visualized in vitro
  using fluorescent resonance energy transfer (FRET)

Zaccolo, M. et al. Nat Cell Biol. 2000
Jan2(1)25-9
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Zaccolo, M. et al. Nat Cell Biol. 2000
Jan2(1)25-9
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Zaccolo, M. et al. Nat Cell Biol. 2000
Jan2(1)25-9
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PKA and transcriptional regulation

• PKA regulates gene transcription in response to
  cAMP
• Glucagon induction of PCK1 (rate limiting enzyme
  in gluconeogenesis)
• Requires C? translocation in to nucleus
• Diffusion (passive) through nuclear pore complex
• C has no endogenous NLS
• Phosphorylates transcription factors
• Nuclear export of C? is facilitated by inhibitor,
  PKI
• Not absolutely required to stop transcription

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C?- PKA nuclear translocation is stimulated by
cAMP
(-) dibutyryl cAMP (1 mM)
() dibutyryl cAMP (1 mM)
(-) dibutyryl cAMP (1 mM)
() dibutyryl cAMP (1 mM)
Anti C-PKA immunofluorescence was detected with
anti-rabbit IgG antibody conjugated with
fluorescein (A and B) or rhodamine (C and D).
Ventra, C. et al. J Neurochem 1996
Apr66(4)1752-61
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Evidence that RII can also translocate
Wiley, J. et al. J Biol Chem 1999 Mar
5274(10)6381-7
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CREB

• cAMP responsive element (CRE) binding protein
• Leucine zipper, basic DNA binding domain (bZIP)
• CBP interaction domain
• Kinase-inducible domain
• Ser133 phosphorylated by PKA
• Promotes interactions with CBP

Reviewed by Daniel, P. et al. Annu Rev Nutr
199818353-83
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rCREB dimer phosphorylation induces
conformational change
S133 S133-P S133-P
Usukura, J. et al. Genes Cells 2000
Jun5(6)515-22
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PEPCK promoter

• Highly regulated
• CREB binding elements as well as
• Thyroid hormone, glucocorticoid, isulin, and
  peroxisome proliferator-activated receptor
  responsive elements
• Glucagon synergistically regulates PEPCK with
  glucocorticoids through the co-activator PGC-1

Reviewed by Hanson, R. Reshef, L. Annu. Rev.
Biochem. 66, 581-611 (1997)
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Dominant negative CREB causes hypoglycemia

• A-CREB (dominant neg)
• Acidic extension of leucine zipper
• Disrupts DNA binding of CREB members but enhances
  affinity
• Expression confined to liver
• A-CREB transgenic mice exhibit profound
  hypoglycemia at birth
• Altered expression of CREB regulated enzymes

Herzig, S. et al. Nature 2001 Sep
3413(6852)179-83
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CREB PCG-1 synergism
Herzig, S. et al. Nature 2001 Sep
3413(6852)179-83
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Conclusions

• Cyclic nucleotides regulate a diverse array of
  physiological responses to hormones and growth
  factors
• Responses to increased cAMP are mediated by
  effectors such as PKA, which may also be highly
  regulated
• Gene transcription in response to cAMP requires
  PKA translocation, phosphorylation of
  transcription factors (CREB), and coordination
  with other co-activators and enhancers