Pyruvate Kinase M2 : linking glycolysis to amino acid biosynthesis

The presented work was done in collaboration with Astex Pharmaceuticals

I am a consultant of Astex Pharmaceuticals

I will not discuss off label use and/or investigational use in my presentation

Disclosure Information2012 AACR annual meeting

Eyal Gottlieb

PK

LactatePyruvate

PEP

3PG

Glucose

GA3P

Respiration

G6P

E1PKLR Gene1q21

E2 E3 E4 E5 E12

CAAT TATA

PKRmRNA E1 E2 E3E4E5

PKLmRNA E2 E3E4E5 E6-E12

E6-E12

Pyruvate Kinase L/R isoforms

E10

E9 E10E8 E12E11

E9E8 E12E11 E10E8 E12E11

PKM1 mRNA PKM2 mRNA

PKMgene15q22

Pyruvate Kinase M1/M2 isoforms

B Domain 

A Domain 

C Domain

Active Site 

FBP 

Tyr105 (phosphorylation)

Lys305 (acetylation)

Cys358 (oxidation)

Pro403/ 408 (hydroxylation)

180º

Inter-subunit interface

PKM2

LactatePyruvate

PEP

3PG

Glucose

GA3P

PPP

Serine

Respiration

Glycine

G6P

ROS

Nucleotides

Phospholipids

Cancer cell lines express high levels of PKM2

Silencing PKM2 expression does not change ATP levels and proliferation capacity of HCT116 cells

Metabolic adaptation to PKM2 silencing in HCT116 cells

PKM2 silencing diverts more glucose-derived carbons to serine/glycine biosynthesis and oxidative

phosphorylation

Metabolic adaptation to PKM2 silencing in HCT116 cells

Serine and glycine starvation decreases PK activity

Serine binds to and activates PKM2 in vitro

PKM2 H464A

[compound] (M)

% a

ctiv

atio

n

10- 1 0 10- 8 10- 6 10- 4 10- 2 100

0

1000

2000

3000

4000SerFBP

PKM2 S437Y

[compound] (M)

% a

ctiv

atio

n

10- 1 0 10- 8 10- 6 10- 4 10- 2 100

0

200

400

600

800SerFBP

ITC data PKM2 WT PKM2 H464A PKM2 S437YKd for Ser 200µM N/B 210µM

Mutation of the amino acid binding pocket abolishes serine activation

Serine specifically activates PKM2but not PKM1 or PKLR

PKLR

[compound] (M)

Fold

-act

ivat

ion

(rel

ativ

e to

no

enzy

me

cont

rol)

10- 1 0 10- 8 10- 6 10- 4 10- 2 1000

5

10

15SerFBP

PKM1

[compound] (M)

Fold

-act

ivat

ion

(rel

ativ

e to

no

enzy

me

cont

rol)

10- 1 0 10- 8 10- 6 10- 4 10- 2 1000

5

10

15

20

SerFBP

RLU

Serine 1

0mM

Lysine 1

0mM

Tryptophan

10mM

Tyrosin

e 10m

M

Threonine 1

0mM

Aspara

gine 10m

M

Glutamate

10mM

Basal

activ

ity

Alanine 1

0mM

Arginine 1

0mM

Histidine 1

0mM

Isoleu

cine 1

0mM

Leucin

e 10m

M

Aspart

ate 10

mM

Glutamine 1

0mM

noPEP

Glycine 1

0mM

Prolin

e 10m

M

Valine 1

0mM

Phenyla

lanine 1

0mM

Methionine 1

0mM

Cystin

e 10m

MFBP 50

uMFBP 50

uM

0

200

400

600PKM2

Of all proteinogenic amino acids only serine activates PKM2

Two known modifier amino acids of PKM2 (Tyr105 and Cys358) are in the vicinity of the serine binding domain

Acknowledgements

Marc O’ReillyPetra HillmannAgnès MartinJoe Coyle

Finn HoldingAdam Hold

Barbara ChanetonLiang (Leon) ZhengChristian FrezzaElaine MacKenzie