Developing New Assays for FXM Assay!!!. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH ...

Developing New Assays for FXM

Assay!!!

FXM, 4th Annual AfCS Meeting

UDP C5a IgG2a

Ca2+

Akt-PH

Need to monitor signal transduction

Many unexpected phenotypes from RNAi perturbation experiments


UDP C5a IgG2a

Ca2+

Akt-PH

Phosphoprotein assay IP3 Assay Translocation Assay Others


Pho

spho

prot

ein

tar

gets

sug

gest

ed b

y co

mm

ittee

UDP C5a IgG2a

Ca2+

Akt-PHErk p38

p90

MARCKS

Pyk2

paxillin

p70 S6K

CREB

RiboS6

FKHR

MLC

cPLA2, RiboS6, CREB

Rac

PAK

mTOR

p70 S6K

Pyk2, FAK paxillin

GSK3


UDP C5a IgG2a

Ca2+

Akt-PH

Pyk2

mTOR

p70 S6K

Akt

Erk p38

p90

Pyk2

p70 S6K

Phosphoprotein readout for FXM

2 modest responses Several weak responses


Increased P-Akt response to C5a in Gi2 knockdown cells

Akt (S473)

RhoGDI

NS

C5a

1’

C5a

3’

C5a

10’

C5a

30’

NS

NS

C5a

1’

C5a

3’

C5a

10’

C5a

30’

NS

control Gi2

P-Akt response to C5a

0.00

4.00

8.00

12.00

16.00

0.00 10.00 20.00 30.00 40.00

fold

control

Gai2

(min)

P-p38 response to UDP

0.00

2.00

4.00

6.00

8.00

10.00

0.00 10.00 20.00 30.00 40.00

fold

control

Gai2

(min)


P-Akt response is PTx sensitive

NS

C5a

1’

C5a

3’

C5a

10’

UD

P 3

’

NS

NS

C5a

1’

C5a

3’

C5a

10’

UD

P 3

’

+PTx -PTx

Akt (S473)control

+PTx -PTx

NS

NS

C5a

1’

C5a

3’

C5a

10’

UD

P 3

’

NS

NS

C5a

1’

C5a

3’

C5a

10’

UD

P 3

’

Akt (S473)

Gi2

RA

W 2

64.7

con

tro

l

C5a

R

G

i2

G

q

Anti-Gi2

P-Akt response to C5a

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 2 4 6 8 10 12

fold

control +PTx

control -PTx

Gai2 +PTx

Gai2 -PTx

(min)


Differential effect of PTx on Ca2+ and PIP3 in Gi2 knockdown cells

C5aR

GiG

PI3KPLC

Ca2+ PIP3

Gi2 knockdown

PTx

PIP3

C5aR

GiG

PI3KPLC

Ca2+

control

PTx

Gq


Decreased P-p38 response to UDP in Gq knockdown cells

p38MAPK (T180/Y182)

Pyk2 (Y402)

RhoGDI

NS

UD

P 1

’

UD

P 3

’

UD

P 1

0’

NS

NS

UD

P 1

’

UD

P 3

’

UD

P 1

0’

NS

control Gq

Phosphoprotein response to UDP

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 2 4 6 8 10 12

fold

control p38Gaq p38control Pyk2Gaq Pyk2

(min)

RA

W 2

64.7

con

tro

l

C5a

R

G

i2

G

q

Anti-Gq


Phosphoprotein assay

Continue screening for new phospho-specific antibodies

Develop new technologies: SILAC, AQUA peptide, Bio-Plex

Examine phenotypes in RNAi knockdown cells with FXM P-Ab mixes 1 set of samples for 21 lines replicated samples for 6 lines


UDP C5a IgG2a

Ca2+

Akt-PH



UDP C5a IgG2a

Ca2+

Akt-PHAkt

p38

IP3 PLC

IP3 PI(4,5)P2 PI(3,4,5)P3

PI3K

IP3R

Ca2+

+ +


IP3 Assay

0 50 100 1500

25

50

75

100

125

150

175

200

Time (seconds)

[Ca2

+ ] i(n

M) UDP

IgG2a+ anti-IgG

C5a

Ligand

Macro. Bio. lab

Gain insight on Ca2+ responses to different ligands

Dissect interactions between Ca2+ and PIP3 modules


IP3 Assay

Competitive binding assay Direct measurement Low [IP3], Ca2+/Mg2+ in buffer interferes

with assay

PLC-PH translocation assay Single cell in vivo assay Need development

Accumulation assay Indirect measurement—IP accumulation It works!


UDP C5a IgG2a

Ca2+

Akt-PH



Translocation Assay

GPCR activation and desensitization Arrestin Grk2

Cytoskeleton Actin coronin

Rac/Cdc42 PAK-PBD

PI3K/PIP3 p85 Akt-PH Btk Ship FoxO3

Calcium/PKC PKC CaM-M13 CaMKII Erk NFAT NF-B

Tyrosine kinase Syk

PLC/IP3 PLC-PH


Translocation Assay

Focus on XFP reporters: most constructs are available use ligand cocktail to screen for response

Adopt available FRET assays:


UDP C5a IgG2a

Ca2+

Akt-PH



Other Assays...

Developed: cAMP Lipid macropinocytosis

To be developed: tomorrow’s session

We need robust assays!


How Many Assays Do We need

Estimate based on studies in yeast (Michnick report; G. Yao et al, PLoS Biol. 2 (3), p355-367, March 2004)

~10-20 interacting proteins per signaling step

~200 proteins on FXM parts list

We need 10-20 assays on non-randomly selected targets


How Many Assays Do We need

FXM network:

We need ~10 assays, one for each signaling step

GPCR

GGPLC

FcR

PTK

PI3K

PIP3

IP3

IP3R

Ca2+

Number of assays needed will not increase proportionally as we expand beyond FXM


Acknowledgement

Phosphoprotein assayYan Ni

Audra Wendt

Cell Preparation Lab

Macrophage Biology Lab

Antibody Lab

IP3 assayDianne DeCamp

Meghdad Rahdar

Cell Preparation Lab

Translocation assayMicroscopy Lab

Molecular Biology Lab

Developing New Assays for FXM Assay!!!. FXM, 4 th Annual AfCS Meeting UDP C5a IgG 2a Ca 2+ Akt-PH ...

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