Developing New Assays for FXM
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Transcript of Developing New Assays for FXM
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
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PH
Phosphoprotein assay IP3 Assay Translocation Assay Others
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PH
Phosphoprotein assay IP3 Assay Translocation Assay Others
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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)
FXM, 4th Annual AfCS Meeting
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)
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PH
Phosphoprotein assay IP3 Assay Translocation Assay Others
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PHAkt
p38
IP3 PLC
IP3 PI(4,5)P2 PI(3,4,5)P3
PI3K
IP3R
Ca2+
+ +
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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!
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PH
Phosphoprotein assay IP3 Assay Translocation Assay Others
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
Translocation Assay
Focus on XFP reporters: most constructs are available use ligand cocktail to screen for response
Adopt available FRET assays:
FXM, 4th Annual AfCS Meeting
UDP C5a IgG2a
Ca2+
Akt-PH
Phosphoprotein assay IP3 Assay Translocation Assay Others
FXM, 4th Annual AfCS Meeting
Other Assays...
Developed: cAMP Lipid macropinocytosis
To be developed: tomorrow’s session
We need robust assays!
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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
FXM, 4th Annual AfCS Meeting
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