Figure EV1. T cells from CBLOST and CBLBOST mice develop and function normally. A Structure of the 30 end of the wild-type Cbl allele and of the targeted ...
Molecular Systems Biology
Guillaume Voisinne et al
Assembly dynamics of the CBL and CBLB signalosomes
Expanded View Figures B
A Cbl
Cblb WT
3 ' UTR 13 14
15
3 ' UTR
16
17
18
19 loxP
loxP 3 ' UTR 13 14
15
OST Cblb Cblb OST
16
17
18
7.6
80.1
6.3
52.9
56.4
68.0
1.6
30.0
51.2
104 105
105
36.3 0 103
104
46.6
105
CD45R
I
0
103
52.2
WT CBLOST
103
0
104
105
CD8
J
WT CBLBOST
6
WT CBLBOST
CD4
103
103
104
62.4
105
20
1000 100
D a an nti- 3 ti- CD C 3 D + 28 PI
ti-
C
0
20
tiC D a an nti- 3 ti- CD C 3 D + 28 PI
0
tian
100
10000
an
10
WT CblOST
IL-2 pg/ml
20
an
0
IL-2 pg/ml
4
1000
30
a an nti- 3 ti- CD C 3 D + 28 PI
8
0
12
D
Thymus Spleen Mln
104
C
0
103
ti-
6
20
H
47.8
0
104 105
0
6
WT CBLOST
C
Thymus Spleen Mln
G
Luminescence X10
0
WT CblbOST
40
2.7 0
a an nti- 3 ti- CD C 3 D + 28 PI
50
WT CBLBOST
81.9
CD8
0
100
104 105
CD8
60
Cells X10
6
WT CblOST
F
103
0
CD45R
WT CBLOST
38.9
105
6.9
0
104
an
CD8
103
CD4
103
40.1 0
105
D
104
CBLBOST
Luminescence X10
103
CD4
4.2 0
58.7
0
104 105
105 103
CD5
0
CD4
52.4
104
78.8
0
104 105
6.9
45.5
CD5
35.6
103
CBLOST
Cells X10
80.7
WT 3.8
150
3 ' UTR
Spleen
Thymus
D
WT
E
m TFP1
OST
Spleen
Thymus
Frt
19
OST
C
loxP Ires
Cbl
OSTT
Figure EV1. T cells from CBLOST and CBLBOST mice develop and function normally. A
B
C D E F G
H I J
Structure of the 30 end of the wild-type Cbl allele and of the targeted CblOST allele following homologous recombination and CRE-mediated excision of the loxP-neorloxP cassette. Exons are shown as filled black boxes and numbered. In the CblOST allele, the One-STrEP-tag (OST) is shown in red and the remaining loxP site in orange. Structure of the 30 end of the wild-type Cblb allele and of the targeted CblbOST allele following homologous recombination and FLP-mediated excision of the frt-neorfrt cassette. Exons are shown as filled black boxes and numbered. In the CblbOST allele, the One-STrEP-tag (OST) is shown in red, the IRES-mTFP1 cassette in green, the two loxP sites in orange, and the remaining frt site in blue. Flow cytometry analysis of thymus and spleen from wild-type (WT) and CBLOST mice for expression of CD4 versus CD8 and CD5 versus CD45R. Numbers adjacent to outlined areas indicate percentage of cells. Flow cytometry analysis of thymus and spleen from wild-type (WT) and CBLBOST mice for expression of CD4 versus CD8 and CD5 versus CD45R. Numbers adjacent to outlined areas indicate percentage of cells. Cellularity of thymus, spleen, and pooled mesenteric lymph nodes (Mln) from wild-type (WT) and CBLOST mice. Data are expressed as mean value SEM. Cellularity of thymus, spleen, and pooled mesenteric lymph nodes (Mln) from wild-type (WT) and CBLBOST mice. Data are expressed as mean value SEM. ATP content of CD4+ T cells purified from WT and CBLOST mice and activated for 48 h with PMA and ionomycin (PI) or with plate-bound anti-CD3 (0.3 lg/ml) in the presence or absence of soluble anti-CD28 (1 lg/ml). ATP content is directly proportional to the numbers of proliferating cells in the well and assessed by luminescence. Data are expressed as mean value SEM. ATP content of CD4+ T cells purified from WT and CBLBOST mice and activated for 48 h with PMA and ionomycin (PI) or with plate-bound anti-CD3 (0.3 lg/ml) in the presence or absence of soluble anti-CD28 (1 lg/ml), assessed by luminescence. Data are expressed as mean value SEM. IL-2 in supernatants of WT and CBLOST CD4+ T cells activated as in (G). Data are expressed as mean value SEM. IL-2 in supernatants of WT and CBLBOST CD4+ T cells activated as in (H). Data are expressed as mean value SEM.
Data information: Data in (C-J) are representative of at least three experiments with at least two mice per genotype.
EV1
Molecular Systems Biology 12: 876 | 2016
ª 2016 The Authors
Guillaume Voisinne et al
Molecular Systems Biology
Assembly dynamics of the CBL and CBLB signalosomes
CBLOST
A
Pearson correlation (R)
1 2 3 Rep. Bio. : Rep. Tech. : 1 2 3 1 2 3 1 2 3
1 1
2
2 3 1
3
2
3 1
2
1 3
1
2
2 3 1
3
2
3 1
1
2
3
1
2
2 3 1
3
2
3 1
1
2
3
1
2
2 3 1
3
2
3 1
2
3
1 0.9 0.8 0.7 0.6 0.5
t=30s
t=120s
t=300s
t=600s
Average R = 0.837
Average R = 0.842
Average R = 0.841
Average R = 0.873
t=0s Average R = 0.809
CBLBOST
B
Pearson correlation (R)
Rep. Bio. : Rep. Tech. :
1 1
2 2
1
3 2
1
1 2
1
2 2
1
3 2
1
1 2
1
2 2
1
3 2
1
1 2
1
2 2
1
3 2
1
1 2
1
2 2
1
3 2
1
2
1 0.9 0.8 0.7 0.6 0.5
t=0s Average R = 0.814
t=30s
t=120s
t=300s
t=600s
Average R = 0.774
Average R = 0.818
Average R = 0.752
Average R = 0.732
1010
CBL iRT normalized intensity
C
CBLOST WT
109 108 107 106 105
104 Rep. Tech. : 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 30s 120s 300s 600s 0s 30s 120s 300s 600s 0s 30s 120s 300s 600s Time : 0s Rep. Bio. 1 Rep. Bio. 2 Rep. Bio. 3 1010
CBLB iRT normalized intensity
D
109
CBLBOST WT
108 107 106 105
104 Rep. Tech. : 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 Time : 0s 30s 120s 300s 600s 0s 30s 120s 300s 600s 0s 30s 120s 300s 600s Rep. Bio. 1 Rep. Bio. 2 Rep. Bio. 3
Figure EV2. Assessment of biological and technical variability across samples. A, B For CBL-OST (A) and CBLB-OST (B) samples, the variability between samples corresponding to a given condition of activation was estimated by computing the Pearson correlation coefficient from log-transformed intensities—normalized using indexed Retention Time peptide intensities (iRT Kit; Biognosys)—of all detected proteins for all pairs of technical and biological replicates (denoted as Rep. Tech. and Rep. Bio., respectively). For each condition of activation (i.e. prior to t = 0 s or following TCR engagement for t = 30 s, t = 120 s, t = 300 s, and t = 600 s), scatter plots of log-transformed and iRT-normalized intensities for all pairs of technical and biological replicates are represented along with the corresponding Pearson correlation coefficients (R). The average Pearson correlation coefficient across all pairs of technical and biological replicates (denoted as Average R) is also indicated for each time point. Strong correlations exist between technical replicates corresponding to the same biological replicate, thereby illustrating the reproducibility of the LC-MS measurement. C, D The iRT-normalized intensity of the CBL (C) or CBLB (D) bait proteins is represented for all the LC-MS runs corresponding to CBL-OST, CBLB-OST, and wild-type (WT) backgrounds. In each sample, a strong enrichment of the bait protein is observed in AP-MS purifications performed in the CBL-OST or CBLB-OST backgrounds as compared to control purifications performed in the WT background. In the case of control purifications from WT backgrounds, missing values are not represented.
ª 2016 The Authors
Molecular Systems Biology 12: 876 | 2016
EV2
Molecular Systems Biology
Guillaume Voisinne et al
Assembly dynamics of the CBL and CBLB signalosomes
A KARS
HIST1H1A HIST1H1C
CBX3
GRAP
EPRS
RARS
ligase activity, forming aminoacyl-tRNA and related compounds
PIK3R1 PIK3CD
proteasome complex
PCB PCCB MCCC2 ligase activity, ACACA forming MCCC1
GRAP2
PIK3CA GRB2
PSMB8
PLCG1 CBLB immune UBASH3A response-regulating CSK signalling pathway
PSMD1
PSMD12
PSMC6
PSMB10 PSMC5 PSMD3
regulation of lymphocyte CD5 activation LGALS1
CAPZA2
CAPZA1 F-actin capping protein complex
FYN
INPP5D
ITGB2 C1QBP
UQCRC2
antigen processing
TUBB
ATP5H
COX4I1
hydrogen ion transmembrane transporter activity
MHC protein binding
RPS15A RPL21 RPL10 DDX3X cytosolic YWHAB ribosome RPS9
UB
TAPBP GNB2
HSPD1 TUBB4B
ANXA6
CLTC CCT8
CD2AP
actin filament
SH3KBP1 IQGAP1
cell-substrate YWHAG YWHAE adherens junction YWHAZ YWHAQ
ARPC1B
unfolded protein binding
SH3 domain binding
PTPRC
COX2 ATP5A1
CAPZB
STAT1 regulation of innate immune SAMHD1 response
respiratory chain
UQCRC1
PIK3R2
PIK3CB
PSMD7
carbon-carbon bounds
ITSN2
signaling adaptor activity
CRK
euchromatin
SDHA
CRKL
phosphatidylinositol 3-kinase complex
LARS
coated pit EPS15L1
B
proteasome complex ITSN2 GRAP
CRKL
NCK2
signaling adaptor activity
GRB2
PSMD6 PSMD13 LAX1 LAT GRAP2
T cell costimulation CD5
UBASH3A
PSMC5
RPS13
PSMD2
antigen processing
HSPA9 RPS19
cytosolic ribosome
antigen receptormediated signaling pathway
RPS20
RPS15A
RPL38
RPS17 RPS14
CSK UB
RPL22
RPS12
RPL30 RPL37A
cell-substrate adherens junction
HSPA8
PFN1
RPS16 RPLP1 RPL23
Figure EV3. GO term enrichment analysis of CBL and CBLB signalosomes. A, B Organization of the CBL (A) and CBLB (B) signalosomes according to GO annotations. Enriched GO terms were extracted from the list of interacting partners identified in this study using the ClueGO plugin (http://apps.cytoscape.org/apps/cluego) on Cytoscape.
EV3
Molecular Systems Biology 12: 876 | 2016
ª 2016 The Authors
Guillaume Voisinne et al
Molecular Systems Biology
Assembly dynamics of the CBL and CBLB signalosomes
B CBLOST HIST1H1A ITGB2 PHGDH RARS RPL10 RPS15A RPS9 SLC25A5 STAT1 UQCRC2 HIST1H1C PSMB8 YWHAB YWHAE YWHAG 14-3-3 proteins YWHAH YWHAQ YWHAZ CD5 CRK CRKL CSK DIAPH2 EPS15L1 FYN GRAP GRB2 INPP5D ITSN2 PCB PIK3CA PIK3CB PIK3CD PI3K subunits PIK3R1 PIK3R2 UB
A CBLBOST ATP5D COX4I1 HSPA8 HSPA9 MCCC1 RPS14 RPS20 ANKRD13A EPS15L1 ITSN2 NCK2 PSMC5 PSMD13 PSMD2 PSMD6 UB USP7 CD5 CRKL CSK GRAP GRAP2 GRB2 LAT LAX1 UBASH3A UBASH3B
list of CBLB interactors 1 2 3 4 5
GLIPR2 RPL22 RPL23 RPL37A RPLP1 RPS13 RPS15A RPS16 RPS17
-1 0 1 Pearson R Correlation
list of CBL interactors 1 2 3 4 5 6 7 8
ACACA CAPZA1 CAPZA2 CAPZB CD2AP HDLBP SH3KBP1
-1 0 1 Pearson R Correlation
C1QBP CBLB PLCG1 PSMC5 PSMD1 PSMD12 PSMD3 PSMD7 ANXA6 ARPC1B CCT8 EPRS GPI HMGB2 KARS LGALS1 PSMB10 PTPRC SAMHD1 SDHA
HIST1H3B PFN1 RPL30 RPL38 RPS12 RPS19 SNRPD3
Normalized Recruitment Intensity 1
0 30 120 300 600
0
ATP1B3 ATP5H CBX3 COX4I1 CYB5B FAM162A GNB2 GRAP2 H2-K1 IQGAP1 LARS MOGS MTCO2 RPL21 SNRPB TAPBP UQCRC1 VDAC3
Time (s)
ATP5A1 CLTC DDB1 DDX3X ESYT1 HSPD1 MCCC1 MCCC2 PCCB PPP2R1A PSMC6 PTBP1 TUBB4B TUBB5 TYW1 UBASH3A UBASH3B
Normalized Recruitment Intensity 1
0 30 120 300 600
0
Time (s)
Figure EV4. K-means clustering analysis of the CBL and CBLB signalosomes. A, B Representation of the CBLB (A) and CBL (B) correlation matrix (Rij) partitioned into different clusters using a K-means clustering algorithm. The normalized recruitment intensity to the bait as a function of time is represented for the different interactors grouped into corresponding clusters. Within each cluster, interactors are listed in alphabetical order. Proteins from the 14-3-3 family and PI3K subunits are highlighted within the CBL signalosome.
ª 2016 The Authors
Molecular Systems Biology 12: 876 | 2016
EV4
0 0
10 2
10 3
10 4
10
3
10 5
CD5
CD
CD
# cells
% of max
80
20
uns
CD5lo CD5med CD5hi
100
40
0 1e4
B
60
60
5
50K
80
CD
3.93
100
D4+
2.76 14.8
120
D4
150K
pLCK(Y505) [% of unstim]
FSC-H
200K
100K
140
CD5hi pLCK(Y505) Geo. Mean.
CD5lo
D
104
3+C
C CD5med
3+C
A 250K
Guillaume Voisinne et al
Assembly dynamics of the CBL and CBLB signalosomes
tim
Molecular Systems Biology
40
20
0 0
10
2
10
3
10
4
10
5
3x103
104
3x104
CD5 pLCK (Y505)
Figure EV5. CD5 contributes to control the phosphorylation of the negative-regulatory tyrosine found at position 505 of LCK via CSK. A Expression of CD5 on short-term expanded CD4+ T cells stimulated by cross-linking biotinylated anti-CD3 and anti-CD4 antibodies with streptavidin for 1 min at 37C. The depicted gates define three populations of T cells with different expression of CD5 at their surface (CD5lo, CD5med and CD5hi). Numbers adjacent to outlined areas indicate percentage of cells. B In addition to having been stained with anti-CD5, the cells described in (A) were permeabilized and stained with an anti-pLCK(Y505). The histogram represents the levels of phospho-LCK(Y505) found in the three populations defined in (A) on the basis of CD5 levels. C The upper panel represents the mean ( SD) of the log fluorescence intensity of phospho-LCK(Y505) as a function of the geometric mean fluorescence intensity of CD5. Mean and SD were computed from populations of cells defined using a regular binning of the CD5 expression histogram (lower panel). D Effect of CD5 cross-linking on the phosphorylation of Y505 of LCK. Short-term expanded CD4+ T cells were stimulated with 2 lg biotinylated anti-CD3 plus 2 lg biotinylated anti-CD4 (as in A) in the presence or absence of 2 lg biotinylated anti-CD5 (clone 53-7.3). The intensity of phospho-LCK(Y505) is represented as percent of phospho-LCK(Y505) intensity in the unstimulated condition.
EV5
Molecular Systems Biology 12: 876 | 2016
ª 2016 The Authors