Protein Tyrosine Phosphatases (PTPs)

1
BCH2021, 2006
Protein Tyrosine Phosphatases (PTPs)
Dr. Daniela Rotin Sick Kids (drotin_at_sickkids.ca)
Reading material Tonks, N, Protein tyrosine
phosphatases from gene, to function, to
disease, Nature Reviews Mol. Cell Biol. 7833,
2006
2

• Lectures overview
• 1. PTPs
• General overview
• Activity
• Classification
• Dimerization
• Oxidation
• 2. Receptor PTPs
• LAR family
• CD45
• PTPalpha/epsilon
• 3. Non-receptor PTPs
• PTP.1B
• SHP-1, SHP-2
• 4. Non-classic PTPs
• PTEN

3
Tyrosine phosphorylation and dephosphporylation
Y
In mammals 90 Tyr kinases 107 Tyr Phosphatases
(PTPs)
4
Protein Tyrosine Phosphatases (PTPs)
1. Although originally thought of as
constitutively active enzyme that dephosphorylate
Tyr phosphorylated proteins (opposing tyrosine
kinase activity), it appears that PTPs are
highly regulated, specific and are equally
important as Tyr kinases. 2. Because Tyr kinases
are regarded as promoting cell signaling and
proliferation, it was initially assumed that
PTPs would inhibit signaling and cell
proliferation. This is not true Some PTPs have
inhibitory and some stimulatory roles in
regulating cell proliferation. 3. PTPs are NOT
related to Ser or Thr phosphatases
5
Due to the dependence on the catalytic Cys in
this reaction, PTPs are very sensitive to
oxidation state of the cell (they are inhibited
by oxidation)
6
(No Transcript)
7
(No Transcript)
8
Tools to identify substrates for PTPs Substrate
Trapping
PTPs are rendered catalytically inactive by
mutating the Cys (C215 in PTP.1B) or Asp (D181
in PTP.1B) needed for catalysis. They still allow
binding to substrates but not dephsophorylating
them, hence the substrates become Trapped
(bound) and can then be identified.
9
Classic PTPs
CLASSIFICATION
(RReceptor NRNon Receptor)
Cell Adhesion molecule
10
Other PTPs
(DSPDual Specificity Phosphatase)
11
Substrate specificity
12
(No Transcript)
13
Dimerization of Receptor PTPs (compared to
receptor Tyrosine kinase, RTK)
14
Interactions between Receptor PTPs lead to
inhibition of catalytic activity Intra- or
inter- molecular interactions between the first
and second phosphatase domain. Eg 1) In LAR
family PTPsD2 (catalytically-inactive) binds D1,
and inhibits its catalytic activity 2) In
PTPalpha, CD45 The first catalytic domain (D1)
binds the Wedge region of another D1
domain (homo-dimerization)
LAR family PTPs
Ig(x3)
The inactive second catalytic domain of LAR
family PTPs (eg PTPdelta) binds to and inhibits
the activity of the first catalytic domain (of
eg PTPsigma).
FNIII (x8)
TM
D1
D2
PTPalpha
The first catalytic domain of PTPalpha binds to
the wedge region and inhibits the activity of
the first catalytic domain of another PTPalpha
(both first and second catalytic domains are
active)
TM
Wedge
D1
D2
15
Oxidation of PTP active site H2O2 incubation
with the catalytic domain of PTP.1B leads to
conformational changes. The PTP loop (containing
the signature motif) and the Tyr from the pTyr
binding loop, which are normally buried, flip out
of the active site and become solvent-exposed.
Does this occur in vivo?
16
Regulation of RPTP function by dimerization a
new role for oxidation
H2O2
Oxidation leads to stabilization of the inactive
state of RPTPs promoting S-S bonds of the Cys in
the second catalytic domain (D2)
17
PTPs and cancer
18
(No Transcript)
19
Receptor PTPs
LAR family PTPs Role in development (especially
the nervous system)
20
(No Transcript)
21
Signaling downstream of LAR family PTPs
Role of the extracellular domain -A cell
adhesion molecule
-PTPdelta and LAR Homophilic interactions of
ectodomain promotes neurite growth and axon
guidance -LAR ectodomain( missing FN5 repeat)
binds laminin-nidogen complex (but biological
consequence of this binding is not
known) -PTPsigma Heterophilic interactions
ectodomain binds heparan sulphate proteoglycan
(HSPG) (agrin and collagen XIII) in chick
retinal axons. In muscle, different ligands are
likely important (not HSPG). This binding does
not affect catalytic activity. Ie. Biological
function of ligand binding not completely
understood.
Role of Intracellular domain
-Generally linked to remodeling of the actin
cytoskeleton -Liprins are LAR family interacting
proteins, but are not substrates. Rather, they
serve as scaffold proteins important
for maintenance of the presynaptic zone, and they
bind GRIP and GIT (glutamate receptor interacting
proteins). They are also important at the
post-synaptic zone. -Trio binds LAR. Trio is a
guanine nucleotide exchange factor for Rac1 and
RhoA, which are important for cytoskeleal
remodeling. Trio also binds Focal adhesion kinase
(FAK) and is phosphorylated by FAK. Trio also
binds MIM-B, a binding partner to PTPdelta. -The
tyrosine kinase Abl and its substrate Ena are
involved in axon growth and guidance, and oppose
function of DLAR in Drosophila (flies). LAR
likely dephosphorylates Ena (ie Ena is its
substrate). -LAR and PTPsigma bind to and
dephosphorylates N-cadherin and beta-catenin.
The catenin complex is itself in complex with
actin. ie it connects between cadherin and the
actin cytoskeleton, and is important for axon
growth. Additional roles -LAR binds the
Insulin receptor and dephosphoryaltes it in
vitro, although In vivo this may be a result of
dephosphorylation of the IR substrates IRS-1 and
-2. Hence LAR regulates insulin signaling.
22

• Biological functions of LAR family PTPs
• Mainly studies in the nervous system, and
  examples from flies and mammals are provided in
• the following slides

23
LAR family PTPs regulate muscle synaptogenesis in
embryonic flies
24
LAR family PTPs regulate axon guidance in the
developing retina of flies
25
(No Transcript)
26

• Role of LAR family PTPs in mammalian development
• LAR family members are strongly expressed in the
  developing nervous system as well as in several
• Epithelial tissues (eg. lung, intestine)
• 1.Neuroendocrine PTPsigma regulates Growth
  Hormone and Prolactin secretion from
• the pituitary
• 2. Neuronal PTPsigma attenuates neurite
  outgrowth and nerve regeneration (see next
  slide),
• while LAR accelerates them.
• PTPsigma also regulates axon guidance.
• PTPdelta also involved in regulating CNS function
  (learning and memory)
• 3. Pancreas LAR (and probably PTPsigma)
  involved in regulation of insulin signaling
  downstream
• of the Insulin Receptor and hence blood glucose
  levels (reveal insulin resistance).
• LAR family and cancer
• Not much is known, and mostly correlative data
• (eg increase LAR expression in metastatic breast
  cancer, and PTPsigma reduces colony formation
• (indicative of cancer) in soft agar assay)

27
Acceleration of Neurite outgrowth in PTPsigma
knockout mice
28
CD45 Role in regulation of the immune system
29
(No Transcript)
30
(No Transcript)
31

• Regulation of CD45
• Dimerization
• -Homo-dimerization of CD45 inhibits its catalytic
  activity, thus attenuating T cell activation.
• (mutation in the Wedge sequence of CD45 in mice
  relieves its inhibition of phosphatase activity,
  leading to enhanced CD45 and
• TCR activation, and to lymphoproliferative
  disease and autoimmunity)
• -The tendency to dimerize is greater with the
  CD45RO isoform (which is less glycosylated).

2) Spatio-temporal -CD45 association with lipid
rafts (where the TCR complex is found) is
important for its regulation of TCR activation.
3) Phosphorylation CD45 is phsophorylated (by
CK2, PKC, CSK), but the physiological
significance of this is unknown.
32
PTPalpha, PTPepsilon Regulation of Src signaling
and cancer
33
Regulation of PTPalpha
34
(No Transcript)
35
(No Transcript)
36
PTPalpha (and PTPepsilon) activate Src by
dephosphorylating its inhibitory site
(Tyr527). This leads to enhanced cell
proliferation (ie PTPalpha has a positive role in
regulating cell signaling). It may also have a
role in cancer (reminiscent of v-Src which is
constitutively active).
v-Src (viral Src) (Oncogenic)
c-Src
SH3
SH3
SH2
SH2
SH1(catalytic)
SH1(catalytic)
Y527
Dephosphorylated (activated) by PTPalpha
37

• Biological functions of PTPalpha and PTPepsilon
• Activates Src (or other Src family kinases),
  leading to increased cell proliferation and
• transformation
• Important for integrin-mediated signaling and
  hence cell adhesion/spreading
• Important for hippocampal neuronal migration and
  long term potentiation
• Regulates K channels and the NMDA receptors
• Important for bone homeostasis (PTPepsilon)

38
Non Receptor-PTPs
PTP.1B Role in regulation of metabolism
39

• PTP.1B
• Earlier work had demonstrated that the PTP
  inhibitor Vanadate increased insulin signaling
• PTP.1B is a regulator of insulin action and binds
  the activated Insulin receptor (IR) (see next
  slide)
• PTP.1B becomes Tyr phosphorylated following
  binding to the IR
• PTP.1B dephosphorylates the IR (and possibly
  IRS-1, the IR substrate), hence it is a negative
• regulator if insulin signaling. Ie it regulates
  cellular metabolism, not mitogenesis.
• PTP.1B knockout in mice leads to defects in
  glucose and insulin tolerance
• (increased insulin sensitivity) in muscle and
  liver (but not adipose tissues)
• PTP.1B was shown to regulate obesity by
  regulating Leptins action
• PTP.1B is widely expressed. Localized to the ER,
  but a cleavable C terminus allows it
• to enter the cytosol.
• Because PTP.1B knockout mice show increased
  insulin sensitivity and quick clearance of
  glucose
• from the blood, extensive search is now being
  carried out to identify inhibitors of PTP.1B, in
  order
• to treat type II diabetes and obesity.
• Knockdown of PTP.1B (with antisense
  oligonucleotides) in obese mice (ob/ob) resulted
  in
• alleviation of insulin resistance
• PTP.1B knockout mice are resistant to
  diet-induced obesity(and show enhanced leptin
  sensitivity).
• But, a role of PTP.1B in leptin signaling is not
  yet clear.

40
(No Transcript)
41
SHP-1, SHP-2 (SH2- domain containing PTPs) Role
in immune regulation, signaling and human
diseases Diseases associated with mutations in
SHP-1 and SHP-2 SHP-1 autoimmune
disease SHP-2 Noonan Syndrome (NS), Leukemias,
Leopard Syndrome (NS short stature,
characteristic facial features, heart defects.
Incidence 12000 live birth. 50 of patients
have mutations in SHP-2) LS lentigines (sun
spots), ECG abnormalities, Pulmonic valve
stenosis, deafness, growth retardation)
42
SHP-1
(Primarily expressed in hematopoietic cells)
SH2 SH2 PTP

• - Motheaten mice have a frameshift mutation that
  truncates most of the Shp-1 gene
• (they are thus considered null).
• They die of auto-immune disease primarily
  affecting their myeloid cells
• SHP-1 provides a negative regulatory signals in
  the immune system
• (as oppose to SHP-2, which provides positive
  signal for cell proliferation)
• SHP-1 negatively regulates integrin signaling,
  TNF receptor signaling and EpoR signaling

43
SHP-2
Ssubstrate BPbinding protein
44
SHP-2
SHP-2 is widely expressed in many tissues.
SHP-2 deficiency -In flies (Drosophila)
SHP-2(CSW) affects signaling downstream of
tyrosine kinase growth factor receptors (eg
EGFR, FGFR). SHP-2/CSW plays a positive
(signaling enhancing) role, hence its deficiency
affects normal development -In frogs (Xenopus)
SHP-2 is required for gastrulation and
FGFR-dependent development -In mice SHP-2 null
mice are embryonic-lethal (ie SHP-2 is needed for
mammalian development likely downstream of the
FGFR)
45
Regulation of SHP-2 activity
NSNoonan syndrome JMMLJuvenile myelomonocytic
Leukemia
46
Noonan Syndrome/Leukemia Gain of function
mutations Leopard syndrome Dominant negative
(catalytically-inactive) mutations
47
SHP-2 signaling possible models
b) SHP-2 is recruited by the adaptor Gab1 to
dephsophorylate the inhibitory Tyr on Src (Tyr
527), activating Src, which in turn activates
the Ras/Erk pathway. Alternatively, SHP-2 may
dephosphorylate and inactivate CSK, the Tyr
kinase that phosphorylates (and inactivates)
Src-Y527.
a) SHP-2 dephosphorylates the RasGAP binding
site on the receptor, thus blocking rasGAP and
enhancing Ras/Erk signaling
c) SHP-2 may dephosphorylate and inactivate
Sprouty, the Ras signaling inhibitor (which must
be phosphorylated in order to inhibit Ras).
Another possibility is the SHP-2 interferes with
signaling downstream of Erk
48
SHP-2 Signaling (Cont) detailed explanation
49
PTEN
PDZ binding motif
C2
Phosphatase
1. Although PTEN has the PTP signature motif in
its phosphatase domain, it actually
dephosphorylates Phospholipids 2. PTEN is a tumor
suppressor
50
PTEN catalytic activity
51
(No Transcript)
52

• Role of PTEN as a tumor suppressor
• 1. PTEN inhibits cell survival/mitogenic
  signaling pathways mediated by PI3K and AKT,
• hence inactivating mutations in PTEN are
  oncogenic
• 2. PTEN mutations have been identified in many
  cancers, such as
• brain, bladder, breast, prostate, endometrial
  cancers.
• 3. PTEN mutations associated with several genetic
  diseases that pre-dispose to cancer
• Eg Cowden syndrome (hamartomas-benign growth that
  can develop into malignancy),
• Bannayan-Zonana syndrome, Lhermitte-Duclos disease

• Role of PTEN in development
• 1. PTEN knockout mice are embryonic lethal (ie
  PTEN needed for embryonic development)
• 2. Tissue-specific conditional knockout of PTEN
  in various tissue
• In T cells mice develop lymphoma
• In B cellsnumerous defects, including resistance
  to apoptosis and increased B cell proliferation
• In heart cardiac hypertrophy
• In brain targeting PTEN to the brain (with
  Cre-GFAP, expressed in astrocytes/glia) leads to
• development of seizures, ataxia and death by 29
  weeks of age, with larger brain overall and
• increased size of regions that express the gene.
  PTEN expressed with the Cre-Nestin promoter
• (brain stem cells)leads to death after birth
  with increased brain structures (and its
  dis-organization)
• In mammary glands increased rate of mammary
  gland proliferation, development and breast
  cancer
• In skin hyperplasia, hyperkeratosis and tumor
  formation
• Ie. All these phenotypes point to a role of PTEN
  in normally inhibiting cell proliferation and
• Transformation likely due to inhibition of the
  PI3K/AKT pathway.

53
Summary

• 1. PTPs dephosphorylate pTyr residues (and dual
  specific PTPs also dephosphorylate pSer or pThr.
• Some PTPs also dephosphorylate phospholipids).
  They are highly specific towards their
  substrates.
• 2. Classic PTP comprise of receptor and cytosolic
  PTPs
• 3. PTPs have either negative or positive
  regulatory role in promoting signaling and cell
  proliferation,
• depending on their substrate and signaling
  pathway.
• 4. PTPs are equally important as Tyr kinases in
  the regulation of signal transduction,
  mitogenesis, cell survival,
• development and cancer

54
(No Transcript)