IGP Cell Signaling Section 2004

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IGP - Cell Signaling Section 2004 Monday,
Februrary 9 900-1000 am Lecture 1 Protein
phosphatases Brian E. Wadzinski Department of
Pharmacology Office 424 RRB Email
brian.wadzinski_at_vanderbilt.edu Phone 3-2080

• Readings
• Review Dr. Colbrans lecture (and his recommended
  readings).
• D. Barford (1996) Molecular mechanisms of the
  protein serine/threonine phosphatases. TIBS
  21407-412.
• 3) S. Wera and B. Hemmings (1995)
  Serine/threonine phosphatases. Biochem. J.
  31117-29. More detail in this review.
• 4) N. Tonks and B. Neel (2001) Combinatorial
  control of the specificity of protein tyrosine
  phosphatases. Curr. Op. Cell Biol. 13182-195.

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• REGULATION OF PROTEIN FUNCTION
• Pre-protein regulation
• Different genes
• Transcriptional regulation
• Alternative splicing
• RNA editing
• Translational regulation
• B. Post-translational modifications
• Reversible phosphorylation of proteins (tyrosine
  and serine/threonine residues)
• Glycosylation
• Long-chain fatty acetylation
• Methylation
• Ubiquitination
• C. Proteolysis/protein turnover
• Proteasomal degradation (polyubiquitination)
• Lysosomal degradation
• D. Binding of regulatory molecules

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Reversible phosphorylation of proteins
Cell Response
Cell Response

• Most, if not all, cellular processes are
  regulated by the reversible phosphorylation of
  proteins.
• The level (stoichiometry) of protein
  phosphorylation is determined by the opposing
  actions of protein kinases and protein
  phosphatases (BALANCE of activities).
• Generally, a very dynamic process (even under
  basal conditions).
• Futile cycle? No - provides rapid control of
  protein phosphorylation following subtle changes
  in the activity of the kinase and/or phosphatase.
• Regulation of protein kinases has been
  extensively studied (Colbran lecture). Only
  recently, has the regulation of protein
  phosphatases been studied.
• The activities of both kinases and phosphatases
  are tightly controlled.

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Classification of protein kinases and protein
phosphatases

• Protein kinase superfamily (500 members)
• Protein tyrosine kinases (PTK)
• Dual-specificity protein kinases
• Protein serine/threonine kinases (PKs)
• All protein kinases share some amino acid
  sequence homology (common ancestral gene).
• Protein phosphatase superfamilies (150
  members)
• Protein tyrosine phosphatase (PTP) superfamily
• Classical PTP family
• Dual-specificity phosphatase (DSP) family
• Protein serine/threonine phosphatase (PP)
  superfamily
• PPP family
• PPM family
• PTPs and DSPs are structurally related similar
  mechanism of catalysis.
• PTPs/DSPs exhibit no sequence or structural
  similarity with PPs.
• PPP and PPM family members share very little
  sequence similarity, but crystal structures and
  catalytic mechanisms are strikingly similar
  (convergent evolution).

150
350
110
40
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Identification of substrates for kinases and
phosphatases

• 1. Substrate selectivity (in vitro
  phosphorylation/dephosphorylation
• of substrates using purified enzymes).
• Technical difficulties (enzyme purity, source of
  substrate, efficiency of reaction).
• Physiologic relevance?
• 2. Exploit activators and inhibitors.
• Pharmacological probes of enzyme function and
  structure.
• Affinity purification ligand.
• Potential drugs (e.g. anti-tumor agents,
  immunosuppressants, etc.).
• 3. Alter expression (e.g. overexpress, antisense,
  knock-outs, etc.).
• 4. Determine localization.
• 5. Identify and characterize multi-protein
  complexes containing kinase or phosphatase of
  interest.

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Protein tyrosine phosphatases (PTPs)
Classical PTPs
Non-transmembrane PTPs
Receptor-type PTPs
Dual-specificity phosphatases (DSPs)

• Members of the PTP superfamily are characterized
  by the presence of a signature motif
  (H-C-X-X-G-X-X-R) within the catalytic domain.
• Classical PTPs (pTyr) and DSPs (pSer/pThr).
• PTPs contain one or two conserved phosphatase
  domains (240-250 amino acids).
• Flanking domains regulate catalytic activity
  either directly or indirectly (by providing sites
  of interaction for other regulatory/targeting
  proteins).

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Features of the active site of classical PTPs
PTP1B - a classical PTP

• Substrates identified include the EGF receptor
  (EGFR) and the insulin receptor (IR).
• Characteristics of PTP1B knockout mice 1)
  increased IR autophosphorylation 2) enhanced
  sensitivity to insulin in skeletal muscle and
  liver 3) show protection from diet-induced
  obesity and 4) normal in all other ways tested.
• Together, studies provide strong evidence that
  PTP1B acts as a major negative regulator of
  insulin signaling. Studies also suggest that a
  small-molecule inhibitor of PTP1B might be an
  effective anti-diabetes/obesity agent.

MKP3 - a dual specificity phosphatase

• Highly specific for ERK (phosphorylated on tyr
  and ser residues by MEK).
• MKP3 binds ERK via noncatalytic domain
  interaction activates phosphatase 30-fold.
• MKP3 expression induced in response to ERK
  activation (feedback inhibition).

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Protein serine/theonine phosphatases (PPs)
Historical perspective

• Misconceptions (e.g. promiscuous enzymes).
• Diversity much greater than initially predicted
  (regulatory subunits and interacting proteins).
• Heavily regulated enzymes.
• Tools to study PPs -- only recently available.

Biochemical classification of PPs

• In the early 1980s, Tom Ingebritsen and Phillip
  Cohen described four classes of enzymes that
  could account for most of the ser/thr phosphatase
  activities measured in tissue extracts.

Type 2 protein phosphatases
Type 1 protein phosphatases
PP2C
PP2B
PP2A
PP1
a
a
a
b
Selectivity for ??? subunit of phosphorylase
kinase
No
No
No
Yes
Sensitivity to Inhibitor-1 and Inhibitor-2
Yes (Mg2)
Yes (Ca2)
No
No
Requirement for divalent cations
No
Yes
Yes
Yes
Association with regulatory subunits
Broad
Intermediate
Broad
Broad
In vitro specificity
No
No
Yes
Yes
Sensitive to okadaic acid
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Molecular classification of PP catalytic subunits
PPP Family
PPM Family
PP1 Subfamily PP1 (a, b, g) PP2A Subfamily PP2A
(a, b) PP4 PP6 PP2B Subfamily PP2B (a, b, g) PP5
Subfamily PP7 Subfamily
PP2C (a, b, g, d) Wip CaMKP
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PPP and PPM family members appear unrelated
(based on their amino acid sequence), but their
tertiary structures exhibit similarities

• A central b-sheet structure forms the active
  site surrounded by a-helices.
• Acidic amino acid residues and water molecules
  coordinate two metal ions (bright green).
• Dephosphorylation is thought to be catalyzed by
  metal-activated water molecules that act as
  nucleophiles. Similar enzymatic mechanism --
  convergent evolution.

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All of the PPP catalytic subunits interact with
other cellular proteins
Phosphatase catalytic subunit
Targeting subunits
Inhibitory proteins
Structural subunits
Regulatory subunits

• Free catalytic subunits of PPP family likely
  exist only transiently in cells as they interact
  with a diverse array of other cellular proteins,
  permitting diverse regulatory mechanisms and
  distinct subcellular localizations.
• Diversity of PPPs is achieved primarily through
  the binding partners, rather than having many
  distinct catalytic units (contrast protein
  kinases and PTPs).

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Protein serine/threonine phosphatase 1 (PP1)
I-1
I-2
Ca2

PP2B
PKA
PP1C
I-2
cAMP
active
soluble
PP1C
particulate
neurabin
NIPP
L5
GM
GL
PP1C
PP1C
PP1C
PP1C
PP1C
Spliceosome/ RNA
Targeted to
Glycogen
Cytoskeleton
Myosin
ER
Glycogen metabolism
Neurite outgrowth Synapse morphology
Smooth muscle relaxation
Protein synthesis
Pre-mRNA splicing
Function
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Inhibitor 1 allows for integration of cAMP and
Ca2 signaling
A phosphatase cascade (PP2B PP1)
Ca2
PP2B
I-1

PP1C
P
I-1
PKA
Inactive
cAMP
Other PP1-binding proteins
In this pathway, Ca2 and cAMP signaling are
antagonistic.
Phosphorylation of I-1 by PKA is required for I-1
binding to, and inhibition of, PP1C.

• DARPP32 (dopamine and cAMP-regulated
  phosphoprotein of Mr 32 kDa)
• Neuronal protein that is functionally homologous
  to I-1.
• Important for the control of several neuronal
  processes.

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A common mechanism for PP1C-binding to cellular
proteins
PP1-binding proteins often utilize a conserved
motif for binding PP1C Arg(Lys) -- Val(Ile)
-- Xaa -- Phe PP1C interacting proteins bind away
from the active site.
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Adrenaline regulation of GS and phosphorylase in
muscle
Adrenaline
Glycogen synthase
Active
Glycogen
GPCR
GM
PP1C
GS
Active
AC
Inactive
Phos. b
Phos. kinase
Inactive
PKA
cAMP
P
Glycogen synthase
Inactive
Glycogen
P
I-1
GM
P
P
Active
Phos. a
PP1C
PP1C
Inactive
Phos. kinase
Active
P

• Adrenaline induces phosphorylation of glycogen
  synthase (GS) at Ser 10 and Ser 30-38 inhibiting
  GS however, these sites are not directly
  phosphorylated by PKA.
• Adrenaline regulates GS and phosphorylase a
  activities in part by modulating PP1 activity
  (via PKA-catalyzed phosphorylation of I-1 and GM).

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Regulation of glycogen synthase in the liver
PTP1B
PTEN
Glc-6-P
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Protein serine/threonine phosphatase 2A (PP2A)
PP2A Holoenzyme
Heterotrimeric enzyme ( Holoenzyme)
Cellular substrates
B
C
Cell-surface receptors
Cytoskeletal proteins
A
Metabolic enzymes
Transcription factors
Cell-cycle regulatory proteins
Protein kinases
Mechanisms of regulation
Inhibitor proteins
Post-translational modifications
Regulatory subunits
Interacting proteins
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Post-translational modifications of subunits
P
C
Ub
P
DYFL-CH3
Association with regulatory B subunits
Interaction with other proteins
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Interacting proteins serving as substrates for an
associated PP2A
Interacting protein
PP2A form
Function
Tau ABaC, ABbC PP2A binds to and
dephosphorylates tau assembly and stability
of microtubules. b-adrenergic receptor AB?C PP2
A associates with and dephosphorylates the
desensitized (internalized) b-AR receptor
resensitization. Ca2/CaM-dependent ABaC
PP2A associates with and dephosphorylates
protein kinase IV (CaMKIV) (inactivates)
CaMKIV negative regulatory of CaMKIV
signaling. Raf kinase AC Raf-associated PP2A
dephosphorylates and activates the kinase
positive regulator of Raf signaling.
Interacting proteins modulating the activity of
an associated PP2A
Interacting protein
PP2A form
Function
I1PP2A and I2PP2A
All Non-competitive/heat-stable inhibitors of
PP2A. Viral proteins (small t) AC Binds to and
inhibits AC dimer displaces B subunit. Alpha4 C
Binds to C modulates phosphatase
activity/levels. Casein kinase IIa AC Binds to
AC dimer phosphorylates C and stimulates
phosphatase activity towards MAPKs CKII
deactivation of the MAPK pathway.
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Protein ser/thr phosphatase 2B (PP2B)
calcineurin

• General points
• High expression in the brain.
• Only phosphatase directly regulated by a second
  messenger (Ca2).
• Inhibited by immunosuppressant drugs cyclosporin
  A and FK506 (inhibited only when
  immunosuppressant is bound to cellular
  immunophilin)
• Native form is a heterodimer of the catalytic A
  subunit and the regulatory B subunit.

Catalytic A subunit (a,b,g)

• Regulatory B subunit (different from the PP2A B
  subunits)
• Small acidic protein that binds 4 Ca2 ions via
  EF hand motifs.
• Highly homologous to calmodulin (CaM).
• Permanently associated with the A subunit
  contrast CaM which only binds in the presence of
  Ca2.
• Myristoylated (membrane association).

• Regulation
• In the absence of Ca2/CaM, AID blocks active
  site.
• Full activation requires binding of Ca2 to the
  B subunit and binding of Ca2/CaM.
• Binds to (AKAP79) A-kinase anchoring
  protein-79 more next hour.

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• Calcium-stimulated dephosphorylation of target
  proteins by calcineurin
• I-1 and DARPP-32 (protein phosphatase cascade!).
• Dynamin I (important for endocytosis).
• Nuclear Factor of Activated T cells (NF-AT).

Signaling through calcium, calcineurin, and NF-AT
in different cell types
T Lymphocytes T cell activation increases
intracellular calcium concentrations, which
stimulates PP2B to dephosphorylate the cytosolic
subunit of NF-AT. Dephosphorylated NF-AT
translocates to the nucleus, where it induces
expression of target genes (e.g. IL-2).
Inhibition of PP2B by immunosuppressant drugs
(cyclosporin A CsA) in complex with their
immunophilin (CpH) suppresses T cell activation.