regulation of ptps by reversible oxidationgenomics.unl.edu/rbc_2012/course_files/mon4.pdf ·...

Regulation of PTPs by reversible oxidation

Arne Östman Dept of Oncology-Pathology

Karolinska Institutet Stockholm Sweden

Reversible tyrosine phosphorylation

proliferation

migration

survival

Reversible tyrosine phosphorylation

oncogenes mediators of angiogenesis cancer drug targets

tumor suppressor genes? inhibitors of angiogenesis? cancer drug targets?

PTPs – an enzyme family of 37 strucurally diverse enzymes

Östman, Hellberg, Böhmer, Nat Rev Cancer, 2006

PTPs are involved in regulation signaling triggered by proliferation, cell-cell- and cell-matrix-interactions

PTP inactivation and cancer Inactivating point mutations PTPρ colon cancer LAR colon cancer PTPγ colon cancer Loss of one gene copy DEP-1 breast, colon, lung cancer Promotor methylation SHP-1 lymphomas GLEPP1 lung cancer Östman, Hellberg, Böhmer, Nat Rev Cancer, 2006

PTPs are negatively regulated by reversible oxidation

Major implications:

- Redox environment of cells regulate tyrosine kinase signaling

- ROS induction is a mean of controling PTP function

Knock-down or up-regulation of ROS-producing or scavenging enzymes affect PTP oxidation and growth factor signaling

X

X

reviewed in Östman, 2011

RTKs GPCRs integrins

BCR TCR

ROS

Activation of various cell surface receptors is associated with increased ROS production and PTP inhibition

reviewed in Östman, 2011

In vivo studies support physiological/pathophysiological relevance of PTP/PTEN oxidation in growth factor signaling

- increased PDGFR-induced VSMC proliferation and restenois In PrxII -/- mice

Choi, Nature, 2005

- anti-oxidants reduce PDGFR-induced VSMC proliferation and restenois Kappert, ATVB, 2006

-- increased IR-signaling, and increased PTEN oxidation, in

Gpx1 -/- mice Loh, Cell Met, 2009

-

- Methods to monitor PTP oxidation - Mitochondrial ROS and GF signaling/PTP oxidation - Peroxidized lipids/arachidonic acid metabolites and GF signaling/PTP oxidation

- Differential effects of thioredoxin and glutathione on PTP oxidation - PTP oxidation as an intrinsic component of RTK-mediated transformation

A novel antibody-based method for detection of oxidized PTPs

Persson et al, PNAS, 2004

Copyright ©2004 by the National Academy of Sciences Persson, Camilla et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1886-1891

UV-irradiation induces PTPα oxidation

Persson, PNAS 2004

An MS-application of the oxPTP Abs for global characterization of PTP expression and oxidation

Karish, Cell, 2011

RTK-induced PTP oxidation involves activation of NADPH oxidases and inhibition of peroxiredoxins

Woo, Cell, 2010

RTK-induced PTP oxidation involves activation of NADPH oxidases and inhibition of peroxiredoxins

Potential involvement of mitochondrial ROS in RTK-induced PTP oxidation?

ShcA protein family

p66Shc: – Produces ROS in mitochondria – Regulates insulin signaling

p66Shc KO mice: ◦ 30% increase in life-span ◦ Increased resistance to oxidative stress ◦ Reduced systemic ROS levels ◦ Protected from age- and diet-induced obesity

(Giorgio et. al. 1999, Nature; Trinei et. al. 2002, Oncogene; Napoli et. al. 2003, PNAS; Giorgio et. al. 2005, Science; Pinton et. al. 2007, Science; Berniakovich

et. al. JBC, 2008)

p66Shc is activated by PDGF stimulation

Frijhoff, in prep.

Frijhoff, in prep.

PDGFR phosphorylation is reduced in p66Shc-/- cells

reduced effects of anti-oxidants in p66Shc-/-cells

PDGF-induced PTP oxidation is decreased in p66Shc cells

Frijhoff, in prep.

Frijhoff, in prep.

PDGF-induced migration is decreased in p66Shc cells

RTK-induced PTP oxidation involves mitochondrial ROS, in addition to

activation of NADPH oxidases and inhibition of peroxiredoxins

Lipid peroxides as mediators of PTP oxidation?

Lipid peroxides -fatty acids or phospholipids with a hydroperoxy group - intermediates in arachidonic acid (AA)-derived synthesis of leukotriens and prostaglandins - RTKs induce AA release by activation of phospholipases -GPx4 ko cells display increased levels of peroxidized lipids (Seiler, Mol Cell, 2009)

Lipid peroxides -fatty acids or phospholipids with a hydroperoxy group - intermediates in arachidonic acid (AA)-derived synthesis of leukotriens and prostaglandins - RTKs induce AA release by activation of phospholipases -Scavenging enzymes include GPx4 (Seiler, Mol Cell, 2009)

Study design: - investigate PDGFR signaling and PTP oxidationin Gpx4 ko cells - investigate in vitro effects effects of peroxidized lipids on PTP oxidation

- Investigate effects of AA on PTP oxidation and PDGFR phosphorylation

GPx4 deletion increases cellular PTP oxidation

PTP actvity in total cell lysates

Conrad, Sandin et al PNAS, 2010

Vitamin E pre-treatment

+ -

GPx4 deletion leads to a Vitamin E-sensitive increase in ligand - induced PDGFβR phosphorylation


GPX4 deletion enhances PDGFβR-induced cytoskeletal rearrangements (”ruffling”)


15HPETE (OOH-fatty acid) induces in vitro PTP oxidation in sub/low micromolar concentrations

GST-PTPcd fusion proteins

alkylation of unoxidized

PTPs

ctr H202

HPETE

PVd-mediated conversion of

oxidized PTPs to SO3-form

detection of SO3 forms

by oxPTP Ab

(modified from Groen, Östman, den Hertog, JBC, 2005)


A novel pathway linking AA metabolism and RTK signaling

RTK-induced AA metabolism can contribute to PTP oxidation -peroxidized lipids are acting as PTP oxidants

role(s) of Trx/TrxR and glutathione in reduction of oxidized PTPs

Roles of Trx and glutathione in reduction of oxidized PTPs

Model systems:

PTP1B and SHP2 oxidation in TrxR1-/- cells

(Trx down/Grx up) Mandal, Can Res, 2010

PTP1B and SHP2 oxidation after BSO treatment of cells

(GRx down) Meng, Mol Cell, 2002

PTP1B and SHP2 oxidation after additon of TrxR/Trx/NADPH to cell lysates or IP preparations

PTP1B, but not SHP2, display increased oxidation in TrxR1-/- cells

Dagnell, in prep

% re

duce

d P

TPs

% re

duce

d P

TPs

PTP1B SHP2

wt TrxR1 ko wt TrxR1 ko

SHP2

SHP2, but not PTP1B, display increased oxidation after BSO treatement

Dagnell, in prep

SHP2

- + - +

PTP1B

% re

duce

d P

TPs

% re

duce

d P

TPs

0

5

10

15

20

25

30

35

40

Ctrl 20 μM BSO

PTP1B activity in wt cells

BSO:

0

10

20

30

40

50

Ctrl 20 μM BSO

SHP-2 activity in wt cells

0

10

20

30

40

50

60

0.4 mM H2O2 Trx Invivo0

20

40

60

80

100

120

0.4 mM H2O2 Trx Invivo

% re

duce

d P

TPs

% re

duce

d P

TPs

Addition of TrxR1/Trx to cell lysates restores activity of PTP1B

Dagnell, in prep

TrxR1/Trx - + - +

SHP2 PTP1B

PTP reducing activity of TrxR1/Trx is more prominent in cell lysates than in PTP1B IP preparations

Dagnell, in prep

PTP1B %

redu

ced

PTP

s

TrxR1/Trx added toIPs

TrxR1/Trx added to

cell lysates

no TrxR1/Trx

PTP IB SHP2

TrxR/ Thioredoxin

GRx/GR/ Glutathione

? TrxR

substrate? Thioredoxin substrate?

TrxR/ Thioredoxin

GRx/GR/ Glutathione

?

- antioxidant

ROS-mediated inhibition of DEP-1 is an intrinsic and compulsory component of FLT3-ITD transformation

+ antioxidant

Godfrey et al, Blood, 2012


FLT3-ITD-expressing cells display increased ROS-levels and decreased DEP-1 activity


Trolox-mediated reversion of FLT3-ITD-transformed cells Is dependent on DEP-1 expression

- antioxidant

ROS-mediated inhibition of DEP-1 is an intrinsic and compulsory component of FLT3-ITD transformation

+ antioxidant


Acknowledgements (EU/PTPNET, Swedish Research Council)

Jeroen Frijhoff Marco Giorgio

Åsa Sandin Marcus Conrad Pontus Aspenström

Markus Dagnell Marcus Conrad Elias Arner

Rinesh Godfrey Markus Dagnell Frank D Böhmer

regulation of ptps by reversible oxidationgenomics.unl.edu/rbc_2012/course_files/mon4.pdf ·...

Documents