Onl_Er_MSB_166837_supinfo0001 18..22 - Semantic Scholar

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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