Cyclic Nucleotide Phosphodiesterases:a small cog in a big wheel

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Cyclic Nucleotide Phosphodiesterasesa small cog
in a big wheel

• Paisley University
• 14th March 2003

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Signalling Molecules

• The components of the cells controlling
  mechanisms.
• In disease, these go wrong.
• If we can identify what has gone wrong in a
  particular condition then we can develop methods
  of artificially blocking or activating the
  affected pathway.

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Cyclic Nucleotides

• Cyclic AMP and cyclic GMP are both important
  second messengers, controlling many cellular
  functions, mostly through the action of specific
  families of protein kinases.
• cAMP cell growth, differentiation, cell
  survival, inflammatory processes.
• cGMP colour vision, muscle contraction.
• They are generated through the action of
  cyclases.
• These convert ATP and GTP to the cyclic form.
• Phosphodiesterases (PDEs) reverse this process by
  cleaving the cyclic ring making the molecule
  inactive.

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Why are cyclic nucleotides important?

• Cyclic nucleotides involved in controlling most
  all cellular processes.
• Many disease states modified by cyclic
  nucleotides concentrations.
• Inflammation.
• Neuro-degeneration.
• Tumor progression.
• Reductions in blood flow.

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What are phosphodiesterases

• These enzymes are the sole means of degrading
  cyclic nucleotides within the cell.
• There are three types. Those which specifically
  hydrolyse cyclic AMP, those which specifically
  hydrolyse cyclic GMP and those which can
  hydrolyse both.

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The PDE Superfamily
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Inhibitors

• Non-specific PDE inhibitors such as caffeine
  possess memory enhancing, anti-inflammatory and
  anti-proliferative effects.
• Theophylline has been prescribed for many years
  for a variety of diseases, particularly
  inflammatory diseases such as asthma.

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Phosphodiesterases in disease-PDE 1

• Controlled by interaction with calcium
• Hydrolyses cAMP and GMP.
• Found in all tissues.
• 3 genes each with multiple splice variants.
• Inhibitors suggested as valuable in
• Cancer
• Parkinson's disease

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Phosphodiesterases in disease-PDE 2

• Hydrolyses cAMP and cGMP.
• Activated by cGMP.
• Found in all tissues.
• 1 gene with a number of splice variants.
• Inhibitors suggested as valuable in
• Memory disorders
• Learning enhancement

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Phosphodiesterases in disease-PDE 3

• Hydrolyses cAMP only.
• Inhibited by cGMP.
• Found in all tissues.
• 2 genes each generating one enzyme.
• Inhibitors valuable in
• Diabetes
• Chronic peripheral artery occlusive disease

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Phosphodiesterases in disease-PDE 4

• Hydrolyses cAMP only.
• Many controlling mechanisms identified (Most
  studied PDE family).
• 4 genes, each generating multiple isoforms (22).
• Members of all genes found in all tissues.

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Disease targets for PDE4 inhibitor therapy

• Asthma
• Atopic Dermatitis
• Auto-immune Diabetes
• AIDS
• Allergic Rhinoconjunctavitis
• Auto-immune Encephalomyelitis
• Allograft Rejection
• Cachexia
• Cerebral Ischaemia
• Cerebral Malaria

• Chrons Disease
• Depression
• Multiple Sclerosis
• Osteoarthritis
• Psoriasis
• Restenosis
• Reperfusion Injury
• Rheumatoid Arthritis
• Septic Shock
• Toxic Shock
• Ulcerative Colitis

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PDE4 Inhibitors and target systems for combating
inflammatory responses

• Mast Cells and Basophils - inhibition of
  degranulation and thus histamine and leukotriene
  release.
• Neutrophils - inhibition of phagocytosis,
  superoxide production, degranulation, chemotaxis
  and PAF production.
• Eosinophils - inhibition of superoxide
  production, decreased viability, attenuated
  secretion of cationic proteins and suppressed
  PAF-induced leukotriene production.
• T-lymphocytes - inhibition of IL2 production and
  release of cytokines including IFN-g and GM-CSF.
• Monocytes - inhibition of the production of
  superoxide, arachidonic acid, TNF-a, IL12 and
  inhibited IL2R expression, phagocytosis and PDGF.
• Macrophages- inhibition of phagocytosis and the
  production of superoxide and TNF-a.

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Phosphodiesterases in disease-PDE 5

• Hydrolyses only cGMP.
• Found in all tissues.
• 1 gene expressing 3 known splice variants.
• Inhibitors valuable in
• Congestive heart failure
• Sexual dysfunction

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Phosphodiesterases in disease-PDE 6

• Hydrolyses only cGMP.
• Found only in the rods and cones of the eye.
• 3 gene with a number of splice variants.

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Phosphodiesterases in disease-PDE 7

• Hydrolyses only cAMP.
• Found in most tissues.
• 2 genes with a number of splice varients.
• Inhibitors suggested as valuable in
• T cell controlled inflammatory diseases
• T cell leukaemia

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Phosphodiesterases in disease-PDE 8

• Hydrolyses only cAMP.
• Found in all tissues.
• 1 gene with a number of splice variants.
• Inhibitors suggested as valuable in

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PDEs in disease-PDE 9

• Hydrolyses cGMP.
• Found in most tissues.
• 1 gene with a number of splice variants.
• No idea what inhibitors would be useful for.

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Phosphodiesterases in disease-PDE 10

• Hydrolyses cAMP and cGMP.
• Found in some tissues.
• 1 gene with a number of splice variants.
• Inhibitors suggested as valuable in
• Memory disorders
• Parkinsons

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Phosphodiesterases in disease-PDE 11

• Hydrolyses cAMP and cGMP.
• Found in some tissues.
• 1 gene with a number of splice variants.
• No idea what inhibitors would be useful for.

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Are you bored yet?

• At last count (December 2002) there were 56
  identified phosphodiesterases.
• Why does the cell bother making so many enzymes
  when all they do is break down 2 second
  messengers?

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Why So Many PDEs?

• Specific inhibitor evidence shows different
  families control different cellular processes.
• PDEs can be separated by being expressed in
  different cell types, e.g. PDE6 in the cones of
  the retina.
• PDEs can also be separated within the cell, held
  within discrete compartments.
• The same PDE can also carry out different roles
  within the same cell, depending upon how it is
  regulated.

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Cyclic AMP signalling
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Cyclic GMP signalling pathway
Neurons or Endothelium
GTP
Protein
Nitric Oxide
Guanylyl Cyclase
cGMP
Protein kinase G
PDE5
Protein-P
5-GMP
Lower Ca2
Relaxation of Smooth muscle
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Compartmentalization of Phosphodiesterases

• Cyclic nucleotides are synthesised by cyclases at
  the plasma membrane.
• Diffusion of cAMP signal is tightly controlled in
  the cell.
• cAMP concentrations can be differentially
  regulated in different regions of the cell.
• PDEs are located in distinct sub-cellular
  compartments
• Affects the activation state of PKA. Associated
  with an anchoring protein (AKAP) in discrete
  compartments

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Subcellular Localisation of Rolipram Sensitive,
cAMP-Specific Phosphodiesterases.Jin, S.L.C, et
al (1998) J. Biol.Chem. 273, 19672-19678.

• PDE 4D gene in FRTL-5 Thyroid cells
• Stimulated with Thyroid-Stimulating Hormone (TSH)
• Differential centrifugation, assessed PDE
  activity
• Immuno-fluorescence to detect relocation of PDE

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Fig. 1.   Western blot analysis of the PDE4
expressed in quiescent FRTL-5 cells. Cells were
cultured and made quiescent as described under
"Experimental Procedures." After centrifugation
at 20,000  g, comparable protein amounts from
the soluble (Sup.) and particulate (Pellet)
fractions were loaded on an SDS-PAGE gel. After
transfer to Immobilon membranes, duplicate lanes
were probed with a nonselective PDE4 polyclonal
antibody (K116) and two PDE4D-selective
monoclonal antibodies (M3S1 and F34-8F4). The
migration of prestained Mr (MW) markers is
reported to the left of each gel, and the
calculated size of the immunoreactive bands is
reported to the right of each gel. A
representative experiment of the four performed
is reported.
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Fig. 8.   Effect of TSH stimulation on
immunoreactivity for PDE4D isoforms in FRTL-5
cells. Cells were cultured, treated with TSH, and
processed for immunocytochemistry as described
under "Experimental Procedures." TSH stimulation
conditions were 0 min (panels A and B), 15 min
(panels C and D), and 24 h (panels E and F). The
left hand column of pictures (panels A, C, and E)
shows the immuno-reactivity obtained using the
PDE4D-specific monoclonal antibody the right
hand column (panels B, D, and F) demonstrates the
blockage of the signals after pre-absorption of
the antibody with the fusion protein. The arrows
denote examples of staining below the plasma
membrane (A) in the perinuclear region (A, C, and
E), on filamentous structures (A and C), and in
the soluble compartment after 24 h TSH
stimulation (E).
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Myomegalin Is a Novel Protein of the
Golgi/Centrosome that Interacts with a Cyclic
Nucleotide Phosphodiesterase.

• Verde, I. Et al (2001) J. Biol. Chem 276
  11189-11198.

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Fig. 6.   Colocalization of myomegalin and PDE4D
in mouse skeletal muscle. Sections of mouse
skeletal muscle were stained with myomegalin
(PBP4, red) and PDE4D-specific (M3S1, green)
antibodies. The distance between the two
contiguous bands was estimated to be 2 µm,
consistent with the length of a sarcomere. The
staining with myomegalin and PDE4D antibodies
overlapped with that of desmin but not with
either myosin or actin. The periodic pattern of
PDE4D staining was confirmed by a second
polyclonal anti-PDE4 antibody. In all instances,
staining could be blocked by preadsorption of the
primary antibody with the corresponding peptide
or fusion protein. Similar results were obtained
with rat skeletal muscle.
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Phosphodiesterase 4D and Protein Kinase A Type II
constitute a Signalling Unit in the Centrosomal
Area.

• Tasken, K.A., et al (2000) J. Biol. Chem 276
  11189-11198.
• mAKAP assembles a protein kinase A/PDE4
  phosphodiesterase cAMP signalling module.
• Dodge K.L., et al (2001) EMBO J 20(8) 1921-1930.

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Differential regulation of phosphodiesterases.
CATALYTIC
REGULATORY
REGULATORY

• PDEs are multi-domain proteins
• Catalytic unit shows homology between PDE classes
• Specific domains confer sensitivity to regulation
  by other agents and for intracellular targeting.
• UCR regions uniquely characterise PDE4 isoenzymes

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Activation of recombinant PDE4D3 by PKA
Taken from Sette Conti (1996) Biochem 271, 16526
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PDE4D3 exhibits a site for phosphorylation by ERK
(MAP kinase).
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Challenge of intact COS1 cells with EGF causes
the ERK2-mediated phosphorylation of PDE4D3

• Autoradiograph of PDE4D3 from 32P-labelled COS1
  cells.
• EGF-stimulated phosphorylation is blocked by the
  MEK inhibitor PD98059 and is not seen with the
  ser579ala mutant

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Activation of ERK by EGF
EGF receptor
Grb2
SOS
ras
raf
MEK
PD98059
Erk
PDE4D
p90rsk, PP1G, Elk-1, SAP-1, c-myc, CREB
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ERK2 association with PDE4D3 leads to maximal
phosphorylation.
Wt-4D3
Dbl. mutant

• ERK2 Western blot of PDE4D IP, demonstrating ERK
  association with PDE4D3.
• ERK2 phosphorylation of PDE4D3 Wt. and ERK
  binding mutants.

c
IP
S/n
IP
S/n
Mut- FQF
Mut- KIM
D3

nk
nk
nk
k
k
k
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Challenge of COS1 cells with EGF causes the
transient inhibition of PDE4D3.

• EGF activates erk2 in COS1 cells.
• EGF causes the transient inhibition of PDE4D3 in
  transfected COS1 cells.
• This action is blocked by-
• (I) the MEK inhibitor PD98059
• (ii) mutation of ser579 in the erk consensus site
  of PDE4D3

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The transience of ERK2 inhibition of PDE4D3 in
COS1 cells is ablated upon loss of PKA action.

• Normally, EGF transiently inhibits PDE4D3 in COS1
  cells.
• However, stable inhibition is achieved by either
• (i) blocking PKA action with H89 or
• (ii) mutating the PKA phosphorylation sites in
  PDE4D3.

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Erk2 activation allows EGF to increase cAMP
levels in PDE4D3 transfected COS1 cells.

• EGF did not alter cAMP levels in native COS1
  cells.
• EGF increased cAMP levels in PDE4D3 transfected
  COS1 cells.
• MEK inhibition by PD98059 prevented this increase
• No increase in cAMP was seen in cells transfected
  with the erk2 insensitive ser579ala mutant of
  PDE4D3.

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The same PDE enzyme can exist in different
activity states.

• Differential control of one enzyme in a single
  cell type
• PDE4D3 can be activated by phosphorylation by
  PKA.
• PDE4D3 can be inhibited by phosphorylation by ERK.

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Erk inhibition of PDE4D3 may serve a feedback
inhibitory role.
ras
c-raf
PKA
MEK
ERK
PDE4D3
Such actions depend upon- the c-raf form
being expressed in cells PDE4D3 being a major
PDE in cells or controlling an appropriate PKA
compartment.
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• The same controlling mechanisms can effect PDE
  enzymes from the same family in different ways.

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PDE4 activity graphs showing effects of EGF
stimulated ERK phosphorylation on members of the
PDE4D subfamily.
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The roles of UCR1 and UCR2 in mediating the
effects of ERK2 phosphorylation.
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Phosphodiesterases

• Large family.
• Many sub-families.
• Different tissue distribution.
• Different sub-cellular location.
• Associate with many signalling pathways.
• Attractive drug targets if specific inhibitors
  can be generated.

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What is the point ?

• A simple process, the degradation of a cellular
  messenger, has been made extremely complex.
• This is not an accident.
• By having such a vast combination of controlling
  mechanisms, the cell can use the same messenger
  to switch on or off many processes.
• Remember this is just a small part of the cells
  signalling system.
• It is suggested that there are 518 genes encoding
  protein kinases, many able to generate multiple
  isoforms.