Transcript Lecture PPT (updated)

Biochem 503
December 3, 2008
Protein Ser/Thr Phosphatases (PPP family)
Controlling 99% of cellular phosphorylation
I.
II.
III.
IV.
V.
History
Type-1 Protein Phosphatase (PP1)
Type-2A Protein phosphatase (PP2A)
Calcineurin (PP2B) Ca2+/CaM dependent
PP2C or Mg2+-dependent PPase (MPP)
History of Ser/Thr Protein Phosphatases
1950’s 1. PR Enzyme Gerty Cori
glycogen phosphorylase a to b conversion
(phosphorylase phosphatase)
1960’s a lost decade? Ed Fischer students struggle
S. Tamura
Tohoku Univ.
Sendai, Japan
1970’s
2. Glycogen synthase PPase - Tsuiki, Tamura, Kikuchi
C’ville to Sendai connection…3 different enzymes separated
later known as PP1, PP2A and PP2C (MPP)
3. The EtOH ppt method
a grad student mistake becomes “standard”
method - 35 kDa PP-ase (C subunit)
E.Y.C. Lee
U. Miami,
now NYMC
4. Isolation of inhibitor proteins (1976)..the C + I = CI idea of regulators
5. Same phosphatase for every substrate?…….histone, casein, myosin,
tropomyosin, spectrin, ribosomes, enzymes……
“broad specificity” (i.e. non-specific) 35 kDa phosphatase
History of Ser/Thr Protein Phosphatases
1980’s
6. Separation of two phosphatases in 35 kDa fraction (Ingebritsen)
Classification (1983) Type 1 vs. type 2 A, B, C
based on sensitivity to inhibitor proteins
7. Targeting hypothesis (Cohen, 1985) - glycogen-bound PP1
a dimer of [ PP1C plus glycogen-subunit]
R subunits modulate activity and anchor C
8. PP1 binds to multiple different R subunits: GM and MYPT1
myosin phosphatase PP1C plus MYPT1
9. PP2A binds 60 and 55 kDa subunits to form ABC trimer
common AC, different Bs (B55, B56, B72)
NIH + Sendai + Hiroshima
Sir Phillip Cohen
Dundee, Scotland
History
10.
11.
12.
Marc Mumby
UTSW
Quick Time™a nd a
TIFF ( Unco mpre ssed ) dec ompr esso r
ar e nee ded to see this pictur e.
Edgar de Cruz e Silva
Dundee, NYC, Portugal
Cloning of C subunits of PP2A
and PP1
unrelated to PTPs, but similar to purple acid PP-ase
Yeast-fungal genetics show functional conservation GLC7=PP1
Discovery of Okadaic Acid as selective PP1-PP2A inhibitor
1990’s
13.
Cloning of multiple PPP catalytic subunits PP4, PP5, PP6
Family Conservation & Diversity in Catalytic subunits
(see PTW Cohen TIBS 22:245-251)
14. Many PP2A B subunits in Families- B, B’, B”
15. Genomics - over 40 human genes, 24 in yeast
QuickT ime ™an d a
TIFF ( Uncomp res sed) deco mpre ssor
ar e need ed to see this pictur e.
David Virshup
Univ. Utah,
now Singapore
NUS-Duke Med Sch.
B
QuickTi me™ and a
T IFF (Uncom pressed) decom pressor
are needed to see t his pict ure.
Tricia (PTW) Cohen
(Lady Cohen)
Dundee, Scotland
Anna DePaoli-Roach
Indiana U Med.Sch.
The PPP Family of Protein Ser/Thr Phosphatases
Toxin-sensitive PPP
PP1
PP2A
PP4
PP6
CaN (PP2B)
Red = human
Green = Drosophila
Yellow = yeast
Natural Toxins from Diverse Sources
Bind and Inhibit PPP Protein Phosphatases
Blister beetleColeoptera
Blue-green Algae
Dinoflagellates
Prorocentrum lima
Streptomyces (fostreus)
Protein Ser/Thr Phosphatases Are Dominant
over Protein Kinases
most proteins are maintained in a de-phosphorylated state
+ PPP inhibitor
Cell Signaling Technology 2002 catalogue (pg. 15): Western blot analysis of whole cell lysates of Jurkat
cells,.untreated with 0.1M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody.
Catalytic Subunit of Protein Phosphatase-1 (PP1)
Okadaic Acid Binds at the Active Site
of PPP Protein Phosphatases
Mechanism of Phospho-Ester Hydrolysis by PPP Phosphatases:
in-line attack of metal-activated hydroxide,
with triginal bipyramid intermediate and inversion of stereochemistry
protonation of the alcoholic leaving group by active site His
Fe
H O
Zn
O
O
P
O
O-Ser-substrate
H-His
A. Catalytic subunit
1. Bimetal center Fe::Zn, Mechanism of direct hydrolysis
Type-1 Protein Phosphatase (PP1)
1. Bi-metallic active site with Fe and Zn
2. 3D structure - beta sheet and alpha helix clusters
3. Isoforms 
 differences mostly in C terminal, allow specific antibodies



alpha
gamma
delta
NPGGRPITPPRN--SAKAKK
--ATRPVTPPRGMITKQAKK
NSG-RPVTPPRTANPPK-KR
4. Phosphorylation in RPITPPR motif first found in yeast sds22
cyclin-dep kinases (CDK) phosphorylate to inactivate;
reversed by auto-dephosphorylation
5. Toxins - microcystin, okadaic acid, calyculin A bind at active site
(3D structures)
B. Regulatory/targeting subunits for PP1C
1. The pioneer…..glycogen-binding GM
2. The RVxF Motif as primary recognition site
3. Myosin phosphatase MYPT1 as regulator & scaffold
4. Dozens more, >200 total…..phospho regulation
C. Inhibitor phospho-proteins
1. Inh1 and DARPP-32
2. Inh2, Inh4
3. CPI-17
4. Linking kinases to PP1 inhibitioninhibitors target selective R-C complexes
D. Substrates
1. abundant phosphatase,
handles large capacity, abundant substrates…
myosin, lamins, glycogen enzymes, histones
Protein Ser(P)/Thr(P) Phosphatase - PP1
many different regulatory-targeting subunits
with common catalytic subunit
C
PP1

RVSF
C
VXF
Neurabin
glycogen GL GM
metabolism PTG
C
C
dendritic spines
VXF
MYPT1
C
KVKF
GADD34
protein synthesis
myosin - cytoskeleton
Dozens of R/K-VxF subunits
Myosin Phosphatase inhibition by
phosphorylation-dependent
inhibitor protein CPI-17.
PKC
P
MYPT1
CPI-17
Myosin Phosphatase: PP1-MYPT1
First example of an inhibitor specific for a PP1 Holoenzyme
Myosin Phosphatase Inhibitor CPI-17
single residue phosphorylation (not T to D mutation)
condenses conformation and increases potency >2000-fold
Selective Inhibition
of Myosin Phosphatase by CPI-17
Phosphatase Activity (%)
same C subunit isoform bound to different R subunits
100
Glycogen-bound PP1
50
0
MYPT1•PP1
10
9
8
7
[P-CPI-17] (-log M)
6
5
Myosin LC Phosphatase: A complex of PP1C with
MYPT1 Subunit
C subunit bound to specific R subunit
Type-2A Protein phosphatase (PP2A)
A. Catalytic subunit
1. Bimetal center Fe::Zn and catalytic Mechanism same as PP1
2. 3D structure…known in complex with A and in ABC
3. Isoforms  10:1 ratio, essential for development
 DYFLCOOH motif at C terminus conserved
phosphorylation - PTKs, eg. Src, JAK
methylation - PMT and PME, alters subunit association
5. Toxins - MCLR, OA bind at active site.
Differences between PP1 and PP2A in 12-13 loop
Protein Ser(P)/Thr(P) Phosphatase - PP2A
ABC = { [A] Scaffold + [C] Catalytic} + [B] Regulatory
ABC
B
A
Tpd3
HEAT
Helical
repeats
A subunit
C
Pph21
Pph22
Binding of C to A decreases Vmax and alters KM
Protein Ser(P)/Thr(P) Phosphatase - PP2A
covalent modifications of the catalytic subunit
ABC
B
A
S-AdoMet
PMT
B
RTPDYFLCOOH RTPDYFLCOOMe
PME
Tpd3
C
PTK
Tyr307
RTPDYFLCOOX
Tyr kinases
Src, JAK, RTK
O
O-P-O
RTPDYFLCOOX
inactive
self
B. Scaffold and Regulatory subunitsthe ABC’s of PP2A.
1. The Scaffold "A" or PR65 (HEAT repeats) the AC dimer
A and A 85:15, but A specific tumor suppressor
2. Many Bs….are they for dedicated functions?
Yeast Cdc55 and Rts1 - distinct phenotypes,
not complemented (conclusion?)
now 3 B families cloned - more than 15 genes
B B55 
B’ B56 
B’’ B72(130)
3. Tumor antigens, sT, mT, E4orf
replace B subunits to re-direct activity.
4. More and more Bs?, p107, p48, etc. proposed as alternates
Protein Ser(P)/Thr(P) Phosphatase - PP2A
multiple dedicated enzymes on one platform
ABC
B
A
4
TAP42
C
anti - apoptosis
kinase inactivation
CDC55
G-like propeller structure
B’56 P
RDS1
Tpd3
C
B55
PPH21
PPH22
anti-apoptosis
integrin signaling
polarity-Wnt signaling
B’’72 P
sm-t
retinoids
tumor virus
transformation
PP2A can be > 80 different “enzymes”
The B56 Family of PP2A Regulatory Subunits
anti-metastatic
Paxillin binding
Wnt-B56-Dishevelled
D-weiderborst
The 3D Structure of PP2A Trimer AB56C
Nature, Nov. 2006
Side view
Top view
C. Inhibitor proteins for PP2A
1. Inh1(PP2A) Damuni et al
2. Inh2(PP2A) SET protein - important in solid tumors
ceramide “receptor” for PP2A activation
D. Alternative partners
1. A binds HSF2 and PP5
2. C binds alpha4; in yeast part of TOR signaling
E. Substrates
1. Signaling Kinases (MEK, PKB, PI3K, p70S6K, etc.)
2. anti-apoptosis (PKB, Bad, Bcl)
3. integrin signaling - Paxillin B56 deletions & increased invasion
Calcineurin (PP2B)(PPP3)
Ca2+/CaM activated Phosphatase
A. Catalytic subunit
1. C has Fe::Zn active site like PP1, PP2A
2. Suppressor domain, CaM dependent
B. Regulatory subunits
1. B subunit (Ca2+)
2. additional CaM
3. FKBP + FK-506 or cyclosporin ($B product)
C. Inhibitor Proteins
1. induced in response to signaling
have reported activity as activator-inhibitor.
D. Substrates
1. NFAT (co-transport into nucleus)
2. Elk-1 (TCF transcription factor)
3. DARPP-32 (NMDA receptor)
Calcineurin (CnA +CaB) Inhibited by
Cyclosporin (CsA) and its Binding protein (Cyp)
$B drug for
immunosuppression
following organ transplants
PP2C = Mg2+-dependent PPase (MPP)
A. Catalytic subunit
1. unrelated to PPP but bimetallic Mg:Mg active center
2. isoforms , etc.
3. many new family members in genome
B. Regulatory subunits - none?
C. Inhibitor Proteins - none?
D. Substrates
1. CDKs
2. the kinase activation loop
3. PI3K
4. Glycogen synthase
Maybe activated by small molecule second messenger? Lipids?
Protein Phosphatase 2C
Mg2+-dependent Phosphatase (MPP)
Protein Phosphorylation
rapid and reversible biochemical reactions
KINASES
ATP
ADP
TARGET
Pi
TARGET
P
H2O
PHOSPHATASES
A molecular on/off switching mechanism.
Protein Phosphorylation: Kinases & Phosphatases
Protein Tyr Kinases
KINASE Superfamily
~ 500 enzymes
ATP
TARGET
Protein Ser/Thr Kinases
P-Tyr TARGET
Cys-SH
ATP
TARGET
PPP
P-Ser/Thr-TARGET
Fe::Zn
Mg Mg
MPP
Protein Tyr(P) Phosphatases
Protein Ser(P)/Thr(P) Phosphatases
Yeast
C. elegans
Drosophila
Human
Yeast
C. elegans
Drosophila
Human
5
95
22
56
13
51
19
15
Protein Phosphorylation: Kinases & Phosphatases
ATP
ATP
ATP
ATP
ATP
ATP
KINASE Superfamily
~ 500 enzymes
ATP
ATP
ATP
ATP
ATP
ATP
Cys-SH
Cys-SH
Fe::Zn
Fe::Zn
Cys-SH
Phosphatase families and ensembles
~ 500 enzymes
Fe::Zn
Mg Mg
MPP
Protein Kinases
and
Protein Phosphatases
ancient enzymes essential to
cell signaling
and
cellular regulation
New targets for Pharmaceuticals