supplementary materials for - science signaling · 2009. 12. 17. · fig. s1. generation and...

www.sciencesignaling.org/cgi/content/full/2/102/ra84/DC1

Supplementary Materials for

Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6

Yonathan Lissanu Deribe, Philipp Wild, Akhila Chandrashaker, Jasna Curak, Mirko H.

H. Schmidt, Yannis Kalaidzidis, Natasa Milutinovic, Irina Kratchmarova, Lukas Buerkle, Michael J. Fetchko, Philipp Schmidt, Saranya Kittanakom, Kevin R. Brown, Igor

Jurisica, Blagoy Blagoev, Marino Zerial, Igor Stagljar,* Ivan Dikic*

*To whom correspondence should be addressed. E-mail: [email protected] (I.D.) and [email protected] (I.S.)

Published 22 December 2009, Sci. Signal. 2, ra84 (2009)

DOI: 10.1126/scisignal.2000576 This PDF file includes:

Fig. S1. Generation and characterization of EGFR bait construct. Fig. S2. Integrating EGFR-interacting proteins from multiple data sets. Fig. S3. Validation of EGFR-interacting proteins identified in the MYTH screen. Fig. S4. HDAC6 interacts with EGFR. Fig. S5. HDAC6 interacts with other ErbB family members, but not with mammalian integral membrane transporters and ion channels. Fig. S6. HDAC6 overexpression increases the abundance of EGFR. Fig. S7. HDAC6 and EGFR partially colocalize at the plasma membrane, and in a fraction of intracellular vesicles following EGF stimulation. Fig. S8. Downregulation of HDAC6 changes characteristics of endosomes. Fig. S9. HSP90 inhibition has no discernible effect on ligand-induced degradation of EGFR. Fig. S10. Identification of tubulin Lys40 acetylation by mass spectrometry. Tables S1 to S7. Annotation of EGFR-interacting proteins identified in MYTH and comparison to prior studies. Movie S1 Legend. Live cell imaging of A431 cells stably expressing GFP-Rab5. References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/2/102/ra84/DC1)

Movie S1 (.mp4 format) NAViGaTOR file for Figure 1A (.xml format)

Supporting Figure S1

E

3 4

220

148

53

30

Anti-VP16

Anti-Gpd1 Anti-Wbp1

220

148

1 2

Anti-VP16

53

30

Cytosolic Membrane

D

MF -EGFR-C-T - +

Anti-VP16

Anti-phospho-

EGFR Y1045

Anti-GPD-1

F

Fur4-NubG

Fur4-NubI

Ost1-NubI

Ost1-NubG

Bait:

MF

-EG

FR

-C-T

Control Preys: SD-Trp-Leu-Ade-His

+ 25 mM 3-AT X-Gal test SD-Trp-Leu

B

X-Gal test

THY AP4 +

MF -EGFR -C-T

THY AP4 +

positive control

THY AP4 +

EGFR-C-T

SD-Leu

C

A

Anti-VP16

250 kDa

148

98

64

53

30 20 14

2 3 1

* * *

no bait

MF -EGFR-C-T

DIC Merge DAPI -VP16

Fig. S1. Generation and characterization of EGFR bait construct. (A) Self-activation test of the EGFR bait

constructs. THY.AP40 cells were transformed with a positive control vector (pLexA-VP16), as well as the

two EGFR bait constructs, EGFR-C-T and MF -EGFR-C-T, and streaked out on SD-Leu and X-Gal plates to

select for a presence of a bait plasmid. Only the MF -EGFR-C-T construct is not self-activating as can be

seen from a lack of blue color on the SD-Leu + X-Gal plate in the absence of an interacting prey construct.

(B) Western blot of total yeast protein extracts. THY.AP40 cells were transformed with empty bait vector

pMF -AMBV (lane 1), EGFR-C-T construct (lane 2), or MF -EGFR-C-T construct (lane 3). Total protein

extracts were prepared, run on an 8% SDS-PAGE gel, and probed with a polyclonal antibody to VP16 . A

single band representing the full-length EGFR-C-T fusion protein at approximately 180 kD is seen only in

the case of the MF -EGFR-C-T construct and no degradation is seen as with the original, EGFRC-T

construct. Asterisk (*) in lanes 1-3 indicates the presence of unspecific proteins. (C) MF -EGFR-C-T

localization in yeast. Fluorescence images were taken by confocal microscopy to show the localization of the

MF -EGFR-C-T bait in yeast. Yeast cells expressing the MF -EGFR-C-T bait or empty bait vector were

fixed, and acetone-treated spheroblasts were probed with rabbit antibody against VP16 and visualized by

Cy3-conjugated antibody against rabbit IgG (red). Nuclei are stained with DAPI (blue). The merged image

illustrates the location of nuclei and confirms the localization of the MF -EGFR-C-T to the yeast plasma

membrane. (D) The modified MF -EGFR-C-T bait protein is properly inserted into the membrane.

THY.AP40 cells were cotransformed with the modified bait construct, MF -EGFR-C-T, and the testing

vectors as listed (Fur4-NubG or NubI and Ost1-NubG or NubI). Growth was assayed by spotting three

independent transformants on selective and nonselective media. Lack of growth on SD-Trp-Leu-Ade-His and

X-Gal plates with two different yeast integral membrane proteins fused to NubG confirms that MF -EGFR-

C-T is not self-activating. Growth on the same plates with NubI constructs indicates that the modified

construct is expressed and the protein is inserted into the membrane as a type I receptor. (E) MF -EGFR-C-T

bait protein is localized within the yeast membrane. Yeast lysates harboring either an empty vector (lanes 1

and 3) or the MF -EGFR-C-T bait construct (lanes 2 and 4) were subjected to two consecutive

ultracentrifugation steps to segregate the cytosolic (lanes 3 and 4) from the detergent-soluble membrane

fractions (lanes 1 and 2). An equal volume from each supernatant was used for the detection of the bait

protein with a polyclonal antibody against VP16 in Western blots. The localization of the endogenous yeast

ER membrane protein Wbp1p to membrane fraction and the cytosolic GPD-1 to cytosolic fraction validates

the centrifugation procedure. (F) MF -EGFR-C-T bait protein is not phosphorylated in yeast. Lysates from

yeast transformed with either an empty vector (lane 1) or MF -EGFR-C-T bait construct (lane 2) were

subjected to Western blotting and probed with an antibody that recognizes EGFR phosphorylated on one of

the key residues phosphorylated upon activation (Tyr1045) to determine if the receptor is phosphorylated in

yeast. Blotting with an antibody against Gpd1p served as a loading control.

HSPCA

HSPD1

INPPL1

DKFZp434N101

LRRC17

CAMLG

RUSC2

CTNND1

DYNC1H1

PTK6

ANTXR1

AMH

PIK3R2

KRT6AJAK2

CENTD2

VIL2

PRKCD

GIT2KRT14

STAT5B

TLN1

GAB1

PIK3R3

ANKS1A

CAV1

LRRC59

HSP90B1

ARHGEF7

PIK3C2A

TUBB

PDCD6IP

RAB3A

CFL1

IRS4

CTNNB1

CTNNA1

VAV1

NCK1

ACTA2

GAB2PLCH1

LIMD1

HSPA5

HSPA1A

SLC25A3

AP2B1

ARF6

UCHL1

CDC25A

HSPA4

ERBB2

CASP1

CST3

PIK3R3

Adapter

FKBP4

Fibronectin

TPI1

YWHAQ

TUBA3

TRIM29

CLTA

NME2 INPP5B

DOK5

PXN

IRS1

TNS4

VAV2

PTPN1

DOK6

DSG2

MEGF6

SNRPD2

DNAJA1

STAM2

CDH1

RALY

PRKAR1B

EFNB1Transforming

CMTM8

PIN4

DOK4

EEF1GPTPN12

AR

NDN

ITLN2

PLD1

ANXA2

CSRP1

PLEC1

ADRM1

BCAR1

CAV2

PIK3C2B

SH3BGRL3

NUMB

MAPK8IP2

HNRPK

PKP2

AHSA1

S100A9

SYK

ZAP70 YWHAE

HGS

TBC1D16SRI

MAP3K14

VAPA

ABL2

SHC3

PRKD1

SLA2

MAP2K1

CD82

TLR2

EIF2B3

CSE1L

GRB7

CAV3CSK

TFRC

TBRG4

DNAJA3

HNRPR

COL9A3

MAP4K1

FES

CEBPB

GNAI2

GABARAPL2

GRB14

IQGAP1

OSAP

CDCP1

PRKAR1A

APBB2

ACTR2

PDK3

FER

GRB2 PTPN2

EREG

MAST1

CDNA:

EPHB4

ACTR3

CDH2

SOCS3

OAZ1

ADRBK1

MUC1TXN

PTGDS

RPS27A

NRG1

PLD2

CLTC

CISH

BTC

PREI3

ANXA1

Gamma-catenin.

ATP5C1

AGTR1

ATP2A2

PSMD4

CLTCL1

DAB2

SYN1

SHC1

CDNA

PTPN6

EFHD2

PRKCA

ALCAM

Protein

HNRPF

XPO6

CBLVAV1

DOK2

MAPK14

SGK223

ALDOA

HDAC6

RP11

PITPNA

mist

SHC2

MDH1

APPL1

PIK3R1

ERRFI1

PLSCR1

DNAJA2

HSPA9B

HBEGF

SLC25A5

PKIA

SH2D1A

TLN1 RABGEF1

PTPRS

BCAR3

TNC

ATP5B

MVP

AXL

KRT7

TMCO3CRKL

CRK

AREG

PNPLA2

GRB10

APBA3

CBR1

ESR1

ANKS1B

ARPC4

CALM1

SH2B3

PDCD6IP

SH3BGRL STAT5A

ARPC3

WBP2

ARPC2

ARPC1BSTAT1

STAM

RASA1

RAP1GDS1

RIN1

Heat

VIM

PTPN11

FAS

PRKACA

EPHA2

FAH

RGS16

PEX19

CBLC

S100A7

CCDC50

HSPB1

CNTN2

S100A11

Synaptosomal-associated

STIP1

GOT1

SFN

TJP1

UBE2V2

HNRPH1

HEXIM1

SH2B

SOCS1

DCN

AHNAK

PTK2

SCAMP1

EPS15

MATR3

PLCG2

DEGS1

APBB3

ATIC

SEC13L1

EPB41

SNX6

CEACAM1 APBB2

ARPC5

SH2D3A

OLFM1HNRPA2B1

HECTD1

CDC2

UROD

SPCS2

Heat

CDNA

ITGA5

SOS2

PTK2B

APBB1

DDX3X

SCAMP3

BLK

EEF1A1

VAV2

CD44

AIP

KRT17

EHF

SOS1

KCTD9

FKBP8

GAPDH

GIT1

RPS27A

SLA

TLR4

SNX1

SDCBP

PSMA7

HSP90AB1CBLB

TOM1L1

SPARCL1

SLC3A2

ZNF259

TNK2

SHC1

SHC1

PRCC

TNS4

RUTBC1

LYN

EPPK1

YES1

PRDX1

DKFZp686J1568

HSPA8

STUB1

MET

ABL1KRT8

NCK2

AKAP12

KRT18

ATP1B1

STAT3

ATP1A1

VAV3

XPOT

DNAJC4

KRT5

CKAP4

HIST1H3A

CTG3a

ACP1

EPS8

PRKX

TPM1

AIDA1C

BMX

ERBB3

hnRNP

SNX4

DCTN2

XRCC6

TGFA

EGF

CEND1SNX2

PTPRJ

ICAM1

SH2D2A

CAMK2G

Cytokine-inducible

MAPK1

YWHAZ

HD

FHL2

PAK1

AP2M1

TUBB2C

PLCG1

ERBB4

HOXC10

GSNRIPK1

ATXN10

HSP90AA1

RIN2

KIAA0777

TUBA1

SRC

Tubulin

KPNB1

PDGFRB

NUMBL

SEPP1

HIST1H2BC

ARF4

HIST1H1E

MAP3K12

DKFZp686P15220

KRT1

ndp52MAPK8IP1

CYLD

HSPA9B

GPM6B

DOK1

EGFR

Unmatched

Energy production

Stress and defense

Transport and sensingAmino acid metabolism

Protein fate

Other metabolismTranscriptionTranslation

Cellular fate and organization

Genome maintenanceNovel MYTH interactorsUnique Blagoev et al. andJones et al. interactorsUnique interactors in I2DInteractors in two or more sources

Fig. S2. Integrating EGFR-interacting proteins from multiple data sets. 403 unique EGFR interactions were rendered with NAViGaTor 2.02 (http://ophid.utoronto.ca/navigator). Node color corresponds to the Gene Ontology, as shown in the key. Diamonds represent previously unknown EGFR identified in the MYTH screen, rectangles correspond to interactions solely identified in the combined data sets from Blagoev et al. and Jones et al. (1, 2), small circles represent interactions solely present in I2D version 1.71 (http://ophid.utoronto.ca/i2d), and large circles correspond to interactions ovelapping in two or more of the data sets ( MYTH, Blagoev/Jones, I2D).

yonathanSticky NoteMigrationConfirmed set by yonathan

yonathanSticky NoteMigrationConfirmed set by yonathan

yonathanSticky NoteAccepted set by yonathan

yonathanSticky NoteAccepted set by yonathan

yonathanSticky NoteCompleted set by yonathan

yonathanSticky NoteCompleted set by yonathan

Anti-EGFR

Anti-His

EGFR - + - +

14-3-3 -His + + - +

TCL IP: EGFR

*

Anti-EGFR

Anti-HA

EGFR + + + + - + - +

FKBP38-HA - + - + + + - +

TCL IP: HA TCL IP: EGFR

*

*

C

A


B

Anti-EGFR

Ponceau

C

*

Fig. S3. Validation of EGFR-interacting proteins identified in the MYTH screen. (A) FKBP38 and 14-3-3 interact with EGFR. HEK293T cells were transfected with plasmids encoding for EGFR and hemaglutinin-tagged FKBP38 (FKBP38-HA) (left panel) or histidine-tagged 14-3-3 (14-3-3 -His) (right). Cell lysates were subjected to immunoprecipitation with antibodies against HA, His, or EGFR as indicated. Blots of total cell lysates (TCL) as input and immunoprecipitates were subsequently probed with an EGFR antibody or HA or His epitope tag antibodies. Asterisks (*) label co-immunoprecipitated EGFR and FKBP30-HA in the left panel and his-tagged 14-3-3 in the right panel. Please note presence of some non-specific bands in both blots that do not represent the immunoprecipitated or the co-immunoprecipitated proteins. (B) Affinity purification confirms that GATE-16 interacts with EGFR. A GST-GATE16 fusion protein was purified from bacteria, coupled to Sepharose beads and incubated with the lysate from A431 cells and the affinity purified proteins were analyzed by SDS-PAGE followed by Western blotting with an EGFR antibody. Ponceau staining shows the abundance of the GST or GST-GATE-16 fusion protein. (C) Validation of putative EGFR interactors by coimmunoprecipitation in mammalian cells. Proteins identified by MYTH that bind EGFR were subcloned into the mammalian expression vector pcDNA3-GFP. Each construct contained an internal HA-tag in addition. Constructs were transfected into HeLa cells, which were lysed 48 hrs later, and proteins were immunoprecipitated with an antibody against HA, or anti-GFP antibody in the case of the negative control. Blots were probed with an antibody against GFP to determine the efficiency of the immunoprecipitation and with EGFR antibodies to detect coimmunoprecipitated EGFR. The cotransfected GFP-tagged DsRed served as a negative control. The asterix marks a nonspecific band caused by HA-immunoprecipitation.


A

Anti-EGFR

Anti-FLAG

HDAC6-FLAG + + + +

EGFR - + - +

TCL IP: EGFR

B

C

TCL

Anti-EGFR

Anti-HDAC6

IP

D

Anti-EGFR

Anti-FLAG

TCL IP: FLAG

EGFR + + + + + + + + + +

HDAC6 wt + +

HDAC6 1-840 + +

HDAC6 dN + +

HDAC6 dBUZ + +

HDAC6 DD + +

Fig. S4. HDAC6 interacts with EGFR. (A) FLAG-HDAC6 coimmunoprecipitates with EGFR. HEK293T cells were transfected with FLAG-HDAC6 alone (lane 1) or together with EGFR (lane 2), cells were lysed, and immunoprecipitation was performed with an EGFR antibody. Samples were loaded as total cell lysate (TCL) and immunoprecipitations (IP). Blots were probed first with the antibody M2 agaist to detect coimmunoprecipitated HDAC6, then stripped, and reprobed with an EGFR antibody to confirm the efficiency of the IP. (B) HDAC6 coimmunoprecipitates with a kinase-inactive mutant of EGFR. Lysates from HEK293T cells overexpressing FLAG-HDAC6 and wild-type EGFR or K721A EGFR were subjected to immunoprecipitation with FLAG antibody. Blots were probed with the EGFR antibody, stripped, and reprobed with the FLAG antibody. (C) Endogenous HDAC6 and EGFR interact. A431 cells were lysed and samples were immunoprecipitated with HDAC6 immune serum or preimmune serum. Blot was probed with an EGFR antibody, stripped, and reprobed with an HDAC6 antibody. (D) Mapping of EGFR binding site on HDAC6 in mammalian cells. HEK293T cells transfected with EGFR and FLAG-HDAC6 wild-type or deletion constructs containing the first 840 residues (HDAC6 1-840), lacking the N-terminus (dN,439-1215), lacking the zinc-finger domain (dBUZ, 1-1132), or lacking deacetylase activity (DD,HDAC6-H216A/H611A). Immunoprecipitations with the M2-FLAG antibody were performed and Western blots were probed with an EGFR antibody, stripped, and reprobed with FLAG antibody

Bait:

NaP

i-IIa-C

-T

Bait:

NH

E3-C

-T

Bait:

SLC

9A

9-C

-T

Bait:

MA

P17-C

-T

Fur4-NubI

HDAC6

Fur4-NubG

Fur4-NubI

HDAC6

Fur4-NubG

Fur4-NubI

HDAC6

Fur4-NubG

Bait:

SLC

22A

4-C

-T

SD-Trp-Leu SD-Trp-Leu

-Ade-His

SD-Trp-Leu

-Ade-His + X-gal

Fur4-NubI

HDAC6

Fur4-NubG

Fur4-NubI

HDAC6

Fur4-NubG


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal

Bait:

Mf

-Erb

B4-C

-T

Fur4-NubI

HDAC6

Fur4-NubG

Bait:

Mf

-Erb

B2-C

-T

Fur4-NubI

NubG-HA-Preys

HDAC6

Fur4-NubG

Bait:

Mf

-Erb

B3-C

-T

Fur4-NubI

NubG-HA-Preys

HDAC6

Fur4-NubG


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal


-Ade-His

SD-Trp-Leu

-Ade-His + X-gal

Fig. S5. HDAC6 interacts with other ErbB family members, but not with mammalian integral membrane

transporters and ion channels. The yeast reporter strain THY.AP40 was transformed with the bait

plasmids bearing the MF -ErbB2-C-T, MF -ErbB3-C-T, or MF -ErbB4-C-T fused receptors, or the

MAP17-C-T, NaPi-IIa-C-T, or NHE3-C-T fused mouse transporters, or the SLC989-C-T or SLC22A4-C-

T fused human ion channels. The resulting strains were transformed with Fur4-NubI or Fur4-NubG and

tested on selective plates (SD Leu-Trp-His-Ade- with 3-AT and X-Gal) to confirm bait expression and

verify that baits were not self-activating. To assess the specificity of the HDAC6 interaction, THY.AP40

was transformed with NubG-HDAC6 and respective bait plasmids, and grown on selective media and

media containing X-Gal. HDAC6 interacted with other ErbB family members (ErbB2, ErbB3, and

ErbB4), but failed to interact with the mouse transporters or the human ion channels.



Anti-EGFR

TCL EGFR (100ng) + + - -

EGFR (500ng) - - + +

HDAC6 - + - +

Anti-FLAG

Anti-ERK2

HDAC6

DAPI

EGF-

Alexa555

Merge

0 Min

Zoom

DAPI Merge

10 min

HDAC6 EGF-

Alexa555 Zoom


Fig. S6. HDAC6 overexpression increases the abundance of EGFR. HEK293T cells were transfected with 100 ng (lanes 1 and 2) or 500 ng (lanes 3 and 4) of a plasmid encoding EGFR alone (lane 1 and 3) or with FLAG-HDAC6 (lane 2 and 4). Cell lysates were run on SDS-PAGE and immunoblotted with EGFR and FLAG antibodies. This experiment was performed 2 times.

Fig. S7. HDAC6 and EGFR partially colocalize at the plasma membrane, and in a fraction of intracellular vesicles following EGF stimulation. HeLa cells were serum starved and pulsed with EGF-Alexa555 (red), fixed immediately or chased for 10 minutes and immunostained with HDAC6 (green) antibody following permeabilization. Confocal microscopy images are shown, with images at a higher magnification marked as zoom.

Parental A549

A549 shRNA HDAC6

Mean inte

g inte

nsity

of

EE

A1 v

esic

les


EGF - 45 90 - 45 90 (min)

-GA +GA

TCL

Anti-tubulin

Anti-EGFR


Fig. S8. Downregulation of HDAC6 changes characteristics of endosomes. Knockdown of HDAC6 increases the signal intensity of endosomes. Parental (black) and HDAC6-knockdown A549 (red) cells were serum starved for 16 hours, pulsed for 30 seconds with EGF-Alexa488, and chased for the time points indicated. After fixation and permeabilization, they were stained with EEA1 and LAMP1 antibodies, and DAPI. Four-channel confocal images were acquired and mean integral intensity of EEA1 vesicles was computed with MotionTracker®.

Fig. S9. HSP90 inhibition has no discernible effect on ligand-induced degradation of EGFR. A549 cells were serum starved, cycloheximide treated, and left untreated or treated with geldanmycin (GA) to inhibit HSP90 activity for four hours. EGF was added for the indicated times and then the cells were lysed. EGFR degradation monitored by probing blots with an EGFR antibody. Tubulin is shown as loading control.


Fig. S10. Identification of tubulin Lys40 acetylation by mass spectrometry. The MS/MS spectrum of the doubly-charged peptide DK*TIGGGD (m/z 402.6904, 2+) derived from Asp-N digestion of tubulin is shown. The asterisk indicates that the lysine residue that was acetylated. As a result of this modification, the peptide, as well as its corresponding fragments, containing acetylated residue displayed a mass shift of 42 Da. Fragment ions containing the N-(b-type ions) or C- (y-type ions) terminus are labeled.

Supplementary Table S1. Annotation of the EGFR‐interacting proteins identified in the MYTH screen. The red shading represents statistically significantsupporting evidence, as determined from random permutations (see Materials and Methods). Protein names, Annotations, and Ontology Terms wereobtained from SwissProt (version 51.5). SPID is the SwissProt identifier. Sig. domain pairs is the number of significant domain pairs that support theinteraction (see Materials and Methods). Gene co‐expression was determined as the Pearson correlation coefficient for the genes encoding theinteracting proteins using the human GeneAtlas expression dataset. Semantic similarity was determined for the GO ‘Cellular Component’ tree (CC),‘Biological Process’ (BP), and ‘Molecular Function’ (MF). The Gene Ontology ‘Cellular Component’ terms obtained from SwissProt were used to determine

Co-localization Semantic SimilarityProtein SPID Annotation Ontology Terms Ontology Category

Sig.

Domain

Gene Co-

expressio

Supplementary Table S1. Annotation of the EGFR‐interacting proteins identified in the MYTH screen. The red shading represents statistically significantsupporting evidence, as determined from random permutations (see Materials and Methods). Protein names, Annotations, and Ontology Terms wereobtained from SwissProt (version 51.5). SPID is the SwissProt identifier. Sig. domain pairs is the number of significant domain pairs that support theinteraction (see Materials and Methods). Gene co‐expression was determined as the Pearson correlation coefficient for the genes encoding theinteracting proteins using the human GeneAtlas expression dataset. Semantic similarity was determined for the GO ‘Cellular Component’ tree (CC),‘Biological Process’ (BP), and ‘Molecular Function’ (MF). The Gene Ontology ‘Cellular Component’ terms obtained from SwissProt were used to determineif the two proteins were annotated to a common cellular compartment; the Co‐localization term column reports the GO term describing thiscompartment. These interactions have been deposited in the I2D database (http://ophid.utoronto.ca/i2d).

CC BP MF

AIP O00170 N/A N/A N/A N/A N/A

PSMA7 O14818

PSA7 Proteasome subunit alpha type 7 (EC 3.4.25.1) (Proteasome subunit RC6‐ 1) (Proteasome b it XAPC7)

proteasome complex (sensu Eukaryota)

F ‐ Protein Fate 4 ‐0.010 1.090 0 0

TermNameSPID Annotation Ontology Terms Ontology Category Domain

Pairs

expressio

n

subunit XAPC7).

MEGF6 O75095

MEGF6 Multiple epidermal growth factor‐like domains 6 precursor (EGF‐like domain‐containing protein 3) (Multiple EGF‐like domain protein 3).

calcium ion binding N ‐ Not Matched 39 ‐0.086 0 0 0.677

protein 3).

KIAA0777 O94875O94875 KIAA0777 protein (Fragment).

U ‐ Uncharacterized 23 0.082 0 0 0

O94992 HEXIM1 protein

cytoplasm; nucleus; cyclin‐dependent protein kinase inhibitor a; protein binding; snRNA binding;

HEXIM1 O94992O94992 HEXIM1 protein (Hexamethylene bis‐acetamide inducible 1).

snRNA binding; transcriptional repressor activity; negative regulation of cyclin‐dependent pro; negative regulation of transcription from R

C ‐ Cellular Fate and Organization

N/A 0.282 1.519 4.772 2.229 GO:0005737

cytoplasm; ATPase

AHSA1 O95433AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1 (AHA1) (p38).

cytoplasm; ATPase stimulator activity; chaperone activator activity; chaperone binding; protein folding; response to stress

F ‐ Protein Fate N/A ‐0.098 1.090 2.276 0.677 GO:0005737

ALDOA Fructose‐bisphosphate fructose‐bisphosphate

ALDOA P04075

ALDOA Fructose‐bisphosphate aldolase A (EC 4.1.2.13) (Muscle‐type aldolase) (Lung cancer antigen NY‐LU‐1).

fructose‐bisphosphate aldolase activity; fructose metabolism; glycolysis; striated muscle contraction

E ‐ Energy Production N/A ‐0.003 0 2.513 1.232

UROD P06132DCUP Uroporphyrinogen decarboxylase (EC 4.1.1.37) (URO‐D) (UPD).

uroporphyrinogen decarboxylase activity

N ‐ Not Matched N/A ‐0.053 0 0 1.232

UCHL1 P09936GELS Gelsolin precursor (Actin‐depolymerizing factor) (ADF) (Brevin) (AGEL)

P62158 N ‐ Not Matched N/A 0.097 2.093 5.079 0.677 GO:0005737(Brevin) (AGEL).

ENPL Endoplasmin precursor (Heat

cytosol; endoplasmic reticulum; endoplasmic reticulum lumen; endoplasmic reticulum membrane; microsome; perinuclear region; calcium

HSP90B1 P14625shock protein 90 kDa beta member 1) (94 kDa glucose‐regulated protein) (GRP94) (gp96 homolog) (Tumor rejection antigen 1).

perinuclear region; calcium ion binding; low‐density lipoprotein receptor binding; protein binding; RNA binding; virion binding; anti‐apoptosis; protein transport; response to


9 0.004 0.629 2.276 0 GO:0005737

transport; response to hypoxia; sequestering of calcium ion

DHCA Carbonyl reductase [NADPH] 1 (EC 1.1.1.184) (NADPH‐dependent carbonyl reductase 1)

CBR1 P16152

carbonyl reductase 1) (Prostaglandin‐E(2) 9‐reductase) (EC 1.1.1.189) (Prostaglandin 9‐ketoreductase) (15‐hydroxyprostaglandin dehydrogenase [NADP+]) (EC 1 1 1 197)

carbonyl reductase (NADPH) activity

N ‐ Not Matched N/A 0.086 0 0 1.232

1.1.1.197).

FAH P16930FAAA Fumarylacetoacetase (EC 3.7.1.2) (Fumarylacetoacetate hydrolase) (Beta‐diketonase) (FAA).

fumarylacetoacetase activity; tyrosine catabolism

N ‐ Not Matched N/A 0.146 0 4.265 1.232

GOT1 P17174

AATC Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) (Transaminase A) (Glutamate oxaloacetate transaminase 1).

aspartate catabolismG ‐ Amino Acid Metabolism

N/A ‐0.021 0 4.265 0

synaptic vesicle; GTPaseRAB3A P20336 RAB3A Ras‐related protein Rab‐3A.

synaptic vesicle; GTPase activity; protein binding; neurotransmitter secretion


6 ‐0.096 2.093 3.809 1.232

CSRP1 P21291CSRP1 Cysteine and glycine‐rich protein 1 (Cysteine‐rich protein 1) (CRP1) (CRP).

zinc ion binding N ‐ Not Matched 2 0.107 0 0 0.677

COF1 Cofilin‐1 (Cofilin, non‐muscle

cytoplasm; nucleus; protein binding; actin cytoskeleton organization and C ‐ Cellular Fate and

CFL1 P23528 isoform) (18 kDa phosphoprotein) (p18).

organization and biogenesis; anti‐apoptosis; Rho protein signal transduction


2 0.034 1.519 3.087 0.677 GO:0005737

EEF1G P26641EF1G Elongation factor 1‐gamma

intracellular; protein binding; translation

P Translation N/A 0 089 0 062 3 362 2 229EEF1G P26641g g

(EF‐1‐gamma) (eEF‐1B gamma).g;

elongation factor activity; translational elongation

P ‐ Translation N/A ‐0.089 0.062 3.362 2.229

PRKAR1B P31321KAP1 cAMP‐dependent protein kinase type I beta regulatory

cAMP‐dependent protein kinase complex; cAMP‐d d t t i ki

C ‐ Cellular Fate and N/A N/A 1 090 3 156 0PRKAR1B P31321 kinase type I‐beta regulatory

subunit.dependent protein kinase regulator act; protein amino acid phosphorylation

C Ce u a ate a dOrganization

N/A N/A 1.090 3.156 0

PUR9 Bifunctional purine

ATIC P31939

biosynthesis protein PURH [Includes: Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (5‐aminoimidazole‐4‐carboxamide ribonucleotide

IMP cyclohydrolase activity; phosphoribosylaminoimidazolecarboxamide for;

l b l idM ‐ Other Metabolism 2 ‐0.006 0 1.249 2.305

carboxamide ribonucleotide formyltransferase) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (Inosinicase) (IMP synthetase) (IMP cyclohydrolase) (ATIC)].

nucleobase, nucleoside, nucleotide and nucl

STIP1 P31948

STIP1 Stress‐induced‐phosphoprotein 1 (STI1) (Hsc70/Hsp90‐organizing protein) (Hop) (Transformation‐sensitive protein IEF SSP 3521) (NY‐ REN‐11

Golgi apparatus; nucleus; response to stress

R ‐ Stress and Defence 6 ‐0.031 2.093 2.276 0 GO:0005634

pantigen).

HSPA4 P34932HSP74 Heat shock 70 kDa protein 4 (Heat shock 70‐related protein APG‐2) (HSP70RY).

cytoplasm; ATP binding; response to unfolded protein

M ‐ Other Metabolism 6 ‐0.075 1.090 2.276 0.677 GO:0005737

MDHC Malate dehydrogenase, cytosol; malic enzyme

MDH1 P40925 cytoplasmic (EC 1.1.1.37) (Cytosolic malate dehydrogenase).

cytosol; malic enzyme activity

N ‐ Not Matched N/A ‐0.147 2.093 0 1.232 GO:0005737

PTGDS Prostaglandin‐H2 D‐isomerase precursor (EC 5.3.99.2)

extracellular region; Golgi apparatus; nuclear envelope; rough

d l i i l

PTGDS P41222

p ( )(Lipocalin‐type prostaglandin‐D synthase) (Glutathione‐independent PGD synthetase) (Prostaglandin‐D2 synthase) (PGD2 synthase) (PGDS2) (PGDS) (Beta‐trace protein) (Cerebrin 28)

endoplasmic reticulum; prostaglandin‐D synthase activity; retinoid binding; transporter activity; prostaglandin biosynthesis; regulation of circadian l / k l

M ‐ Other Metabolism N/A ‐0.127 2.093 3.362 1.232 GO:0005634

(Cerebrin‐28). sleep/wake cycle, s; transport

MATR3 P43243 MATR3 Matrin‐3.nuclear inner membrane; structural molecule activity

N ‐ Not Matched 4 ‐0.223 1.519 0 0 GO:0005634

SEPP1 Selenoprotein P precursorextracellular region;

SEPP1 P49908SEPP1 Selenoprotein P precursor (SeP).

selenium binding; response to oxidative stress

R ‐ Stress and Defence N/A 0.119 0 2.276 0.677

OAZ1 P54368OAZ1 Ornithine decarboxylase antizyme (ODC‐Az).

ornithine decarboxylase inhibitor activity; polyamine biosynthesis

M ‐ Other Metabolism N/A ‐0.027 0 5.742 0

PSMD4 26S proteasome non

PSMD4 P55036

PSMD4 26S proteasome non‐ATPase regulatory subunit 4 (26S proteasome regulatory subunit S5A) (Rpn10) (Multiubiquitin chain‐binding protein) (Antisecretory factor 1) (AF) (ASF).

proteasome complex (sensu Eukaryota)

F ‐ Protein Fate 5 ‐0.128 1.090 0 0

) ( ) ( )

TPI1 P60174TPIS Triosephosphate isomerase (EC 5.3.1.1) (TIM) (Triose‐phosphate isomerase).

triose‐phosphate isomerase activity

E ‐ Energy Production N/A 0.055 0 0 1.232

GABARAPL2

P60520

GBRL2 Gamma‐aminobutyric acid receptor‐associated protein‐like 2 (GABA(A) receptor‐associated protein‐like 2) (Ganglioside e pression factor 2) (GEF 2)

cytoskeleton; intracellular; plasma membrane; actin binding; ATP binding; beta‐tubulin binding; GABA M ‐ Other Metabolism N/A ‐0.063 0.629 4.424 4.758 GO:0005886

2P60520

expression factor 2) (GEF‐2) (General protein transport factor p16) (MAP1 light chain 3‐ related protein).

g;receptor binding; protein binding; protein targeting; synaptic transmission

M Other Metabolism / 0.063 0.629 GO:0005886

cell cortex; endosome;cell cortex; endosome; membrane fraction; plasma membrane; ruffle; GTP binding; GTPase activity; protein binding; cell adhesion; cell motility; cortical actin cytoskeleton

ARF6 P62330 ARF6 ADP‐ribosylation factor 6.

cortical actin cytoskeleton organization an; negative regulation of receptor mediated en; positive regulation of actin filament polym; regulation of Rac protein signal transduction;


4 ‐0.031 2.093 5.183 0.677 GO:0005768

protein signal transduction; ruffle organization and biogenesis; vesicle‐mediated transport

CNTN2 Q02246

CNTN2 Contactin‐2 precursor (Axonin‐1) (Axonal glycoprotein TAG‐

integral to plasma membrane; plasma

C ‐ Cellular Fate and 51 0 113 0 629 3 802 0 GO:0005886CNTN2 Q02246

( ) ( g y p1) (Transient axonal glycoprotein 1) (TAX‐1).

membrane; plasma membrane; cell adhesion Organization

51 ‐0.113 0.629 3.802 0 GO:0005886

FKBP4 Q02790

FKBP4 FK506‐binding protein 4 (EC 5.2.1.8) (Peptidyl‐prolyl cis‐trans isomerase) (PPIase) (Rotamase) (p59 protein) (HSP binding

cytoplasm; intracellular; FK506 binding; protein

F Protein Fate 6 0 107 1 090 2 187 2 013 GO:0005737FKBP4 Q02790 (p59 protein) (HSP‐binding immunophilin) (HBI) (FKBP52 protein) (52 kDa FK506‐binding protein) (FKBP59).

g pbinding, bridging; protein folding

F ‐ Protein Fate 6 0.107 1.090 2.187 2.013 GO:0005737

AKAP12 Q02952

AKA12 A‐kinase anchor protein 12 (A‐kinase anchor protein 250 kDa)

cytoplasm; protein kinase A binding; G protein coupled

C ‐ Cellular Fate and N/A 0 069 1 090 2 968 2 013 GO:0005737AKAP12 Q02952

(AKAP 250) (Myasthenia gravis autoantigen gravin).

binding; G‐protein coupled receptor protein signalin Organization

N/A ‐0.069 1.090 2.968 2.013 GO:0005737

PTPN12 Q05209

PTN12 Tyrosine‐protein phosphatase non‐receptor type 12 (EC 3.1.3.48) (Protein‐tyrosine phosphatase G1) (PTPG1).

cytoplasm; soluble fraction; non‐membrane spanning protein tyrosine phos; SH3 domain binding; protein amino acid dephosphorylation

G ‐ Amino Acid Metabolism

20 ‐0.025 1.090 3.156 1.232 GO:0005737

dephosphorylation

PRDX1 Q06830

PRDX1 Peroxiredoxin‐1 (EC 1.11.1.15) (Thioredoxin peroxidase 2) (Thioredoxin‐dependent peroxide reductase 2) (Proliferation‐associated gene protein) (PAG)

cell proliferation; skeletal development


N/A 0.164 0 3.968 0associated gene protein) (PAG) (Natural killer cell‐enhancing factor A) (NKEF‐A).

AHNAK Q09666AHNK Neuroblast differentiation‐associated protein AHNAK

nucleus; nervous system development

N ‐ Not Matched N/A 0.029 1.519 3.520 0 GO:0005634(Desmoyokin) (Fragments).

development

M3K12 Mitogen activated protein

cytoplasm; cytosol; membrane fraction; plasma membrane; protein binding; protein

MAP3K12 Q12852

M3K12 Mitogen‐activated protein kinase kinase kinase 12 (EC 2.7.11.25) (Mixed lineage kinase) (Leucine‐zipper protein kinase) (ZPK) (Dual leucine zipper bearing kinase) (DLK) (MAPK‐upstream

homodimerization activity; protein kinase binding; protein serine/threonine kinase activity; histone phosphorylation; JNK cascade; peptidyl‐serine


N/A ‐0.102 2.093 6.233 3.732 GO:0005737

) ( ) ( pkinase) (MUK). phosphorylation; peptidyl‐

threonine phosphorylation; protein amino acid autophosphorylation; protein kinase cascade

GPM6B Neuronal membrane integral to membrane; GPM6B Q13491

GPM6B Neuronal membrane glycoprotein M6‐b (M6b).

integral to membrane; nervous system development

N ‐ Not Matched N/A ‐0.103 0.629 3.520 0

DCTN2 Q13561 N/A N/A N/A N/A N/A

COL9A3 Q14050CO9A3 Collagen alpha‐3(IX) chain precursor.

collagen type IX N ‐ Not Matched 2 ‐0.042 4.033 0 0

DYNC1H1 Q14204

DYHC Dynein heavy chain, cytosolic (DYHC) (Cytoplasmic dynein heavy chain 1) (DHC1) (Dynein heavy chain 1, cytoplasmic 1).

cytoplasmic dynein complex; ATPase activity, coupled; microtubule motor activity; protein binding; microtubule‐based

t it ti i dl

D ‐ Genome Maintenance

2 ‐0.047 1.317 3.809 1.232

1, cytoplasmic 1). movement; mitotic spindle organization and biogenesis

FKBP8 Q14318FKBP8 38 kDa FK506‐binding protein homolog (FKBPR38) (FK506‐binding protein 8).

intracellular signaling cascade


6 ‐0.101 0 1.965 0

SPCS2 Q15005

SPCS2 Signal peptidase complex subunit 2 (EC 3.4.‐.‐) (Microsomal signal peptidase 25 kDa subunit) (SPase 25 kDa subunit).

U ‐ Uncharacterized N/A ‐0.081 0 0 0

PDK3 [Pyruvate dehydrogenase

PDK3 Q15120

PDK3 [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 3, mitochondrial precursor (EC 2.7.11.2) (Pyruvate dehydrogenase kinase isoform 3).

mitochondrion; [pyruvate dehydrogenase (lipoamide)] kinase; glucose metabolism


3 ‐0.023 2.093 1.456 3.732

UBE2V2 Q15819

UB2V2 Ubiquitin‐conjugating enzyme E2 variant 2 (MMS2) (Enterocyte differentiation‐associated factor EDAF‐1) (Enterocyte differentiation‐

cytoplasm; nucleus; UBC13‐MMS2 complex; protein binding; ubiquitin conjugating enzyme activity; cell proliferation; protein polyubiquitination;


N/A ‐0.050 0 3.156 0 GO:0005737(Enterocyte differentiation promoting factor) (EDPF‐1) (Vitamin D3‐inducible protein) (DDVit 1).

protein polyubiquitination; regulation of DNA repair; regulation of progression through cell cycle

ADRM1 Q16186ADRM1 Adhesion‐regulating molecule 1 precursor (110 kDa cell

integral to plasma membrane; membrane N ‐ Not Matched N/A 0 159 0 629 0 0ADRM1 Q16186 molecule 1 precursor (110 kDa cell

membrane glycoprotein) (Gp110).membrane; membrane fraction

N ‐ Not Matched N/A 0.159 0.629 0 0

HSPA9B Q1HB43Q1HB43 Heat shock 70kDa protein 9B (Mortalin‐2).

ATP binding; nucleotide binding; unfolded protein binding; protein folding; response to unfolded

M ‐ Other Metabolism 15 N/A 0 0 0p

protein

PLCH1 Q29RV9 Q29RV9 Phospholipase C, eta 1.

calcium ion binding; phosphoinositide phospholipase C activity; intracellular signaling cascade; lipid metabolism


70 N/A 0 0 0

Q4QQI8 RAP1 GTP GDPRAP1GDS1 Q4QQI8

Q4QQI8 RAP1, GTP‐GDP dissociation stimulator 1.

binding N ‐ Not Matched 9 N/A 1.317 0 0

Synaptosomal‐associated

Q53EM2Q53EM2 Synaptosomal‐associated protein 25 isoform SNAP25B variant (Fragment).

synaptosome N ‐ Not Matched N/A N/A 0.062 0 0

Heat Q53GZ6Q53GZ6 Heat shock 70kDa protein 8 isoform 1 variant (Fragment).

ATP binding; nucleotide binding; protein folding; response to unfolded protein

M ‐ Other Metabolism 6 N/A 0 2.276 0.677

Protein Q53XV2Q53XV2 Protein phosphatase 5, catalytic subunit.

binding; hydrolase activity; iron ion binding

N ‐ Not Matched 10 0.052 0 0 1.232

Heat Q59EJ3Q59EJ3 Heat shock 70kDa protein 1A variant (Fragment).

protein binding N ‐ Not Matched 6 N/A 0 2.276 0.677

Fibronectin

Q59G22Q59G22 Fibronectin 1 variant (Fragment).

extracellular region N ‐ Not Matched 25 N/A 0 0 0

TPM1 Q59GR8Q59GR8 TPM1 protein variant (Fragment)

structural constituent of cytoskeleton


2 N/A 0 0 0(Fragment). cytoskeleton Organization

HSPCA Q5CAQ6

Q5CAQ6 Heat shock protein HSP 90‐alpha 4 (Heat shock protein 90kDa alpha (Cytosolic), class A member 1).

ATP binding; nucleotide binding; unfolded protein binding; protein folding; response to unfolded protein

M ‐ Other Metabolism 9 N/A 0 2.276 2.013

SYN1 Q5H9B0 Q5H9B0 Synapsin I.synaptic vesicle; ATP binding; catalytic activity; neurotransmitter secretion

M ‐ Other Metabolism 2 N/A 2.093 3.809 0

membrane; voltage‐gated potassium channel

KCTD9 Q5JSM7Q5JSM7 Potassium channel tetramerisation domain containing 9‐like.

complex; ion channel activity; protein binding; voltage‐gated potassium channel activity; potassium ion transport

A ‐ Transport and Sensing

N/A N/A 0.629 2.178 0.677

RP11 Q5VTK7Q5VTK7 Novel transcript

U U h t i d 23 N/A 0 0 0RP11 Q5VTK7Q5VTK7 Novel transcript (Fragment).

U ‐ Uncharacterized 23 N/A 0 0 0

TMCO3 Q6UWJ1

TMCO3 Transmembrane and coiled‐coil domain‐containing protein 3 precursor (Putative LAG1‐interacting protein).

U ‐ Uncharacterized N/A 0.272 0.629 2.155 0

CEND Cell cycle exit and neuronal CEND1 Q8N111

ydifferentiation protein 1 (BM88 antigen).


RUSC2 Q8N2Y8RUSC2 RUN and SH3 domain‐containing protein 2.

U ‐ Uncharacterized 16 ‐0.089 0 0 0

SPARCL1 Q8N4S1Q8N4S1 SPARC‐like 1 (Mast9, hevin)

calcium ion binding N Not Matched 14 0 113 0 0 0 677SPARCL1 Q8N4S1Q ( , )(Proliferation‐inducing protein 33).

calcium ion binding N ‐ Not Matched 14 ‐0.113 0 0 0.677

OSAP Q8TDB4Q8TDB4 Corneal endothelium specific protein 1 (Ovary‐specific acidic protein).

U ‐ Uncharacterized N/A 0.018 0 0 0

PRCC Proline‐rich protein PRCC

PRCC Q92733

p(Papillary renal cell carcinoma translocation‐associated gene protein).

U ‐ Uncharacterized N/A 0.075 0 0 0

AP2M1 AP‐2 complex subunit mu‐1 (Adaptin mu 1) (AP 2 mu 2 chain)

AP2M1 Q96CW1

(Adaptin mu‐1) (AP‐2 mu‐2 chain) (Clathrin coat assembly protein AP50) (Clathrin coat‐associated protein AP50) (Plasma membrane adaptor AP‐2 50 kDa protein) (HA2 50 kDa subunit) (Clathrin assembly

protein binding; transporter activity

A ‐ Transport and Sensing

N/A 0.084 0 0 0.677

) ( yprotein complex 2 medium chain).

NDN Q99608 NECD Necdin.

negative regulation of cell proliferation; nervous system development; regulation of progression


N/A 0.136 0 3.520 0regulation of progression through cell cycle

OLFM1 Q99784 N/A N/A N/A N/A N/A

DNAJC4 Q9NNZ3

DNJC4 DnaJ homolog subfamily C member 4 (Multiple endocrine neoplasia type 1 candidate protein

membrane; membrane fraction; unfolded protein binding; protein folding; F ‐ Protein Fate N/A ‐0.007 0.062 2.276 2.013Q p yp p

number 18) (DnaJ‐like protein HSPF2).

g; p g;response to unfolded protein

/

PNPLA2 Q9NQ61Q9NQ61 Transport‐secretion protein 2.2 (TTS‐2.2) (Fragment).

lipid metabolism M ‐ Other Metabolism N/A 0.009 0 1.249 0

VAPA Vesicle‐associated membrane protein‐associated protein A (VAMP‐

signal transducer activity; membrane fusion; positive

C ‐ Cellular Fate andVAPA Q9P0L0 associated protein A) (VAMP‐A)

(VAP‐A) (33 kDa Vamp‐associated protein) (VAP‐33).

; pregulation of I‐kappaB kinase/NF‐k; protein complex assembly


N/A 0.023 0 5.285 2.272

cytoplasm; histone deacetylase complex;

HDAC6 Q9UBN7HDAC6 Histone deacetylase 6 (HD6).

deacetylase complex; microtubule; nucleus; histone deacetylase binding; specific transcriptional repressor activity; tubulin deacetylase activity; zinc ion binding;


N/A 0.017 1.519 3.156 2.013 GO:0005737

activity; zinc ion binding; cell cycle; development; histone deacetylation

STUB1 STIP1 homology and U box‐containing protein 1 (EC 6.3.2.‐) (STIP1 homology and U‐box‐

ubiquitin conjugating enzyme complex; Hsp70 protein binding; Hsp90 protein binding; protein binding, bridging; SMAD

STUB1 Q9UNE7

(STIP1 homology and U‐box‐containing protein 1) (Carboxy terminus of Hsp70‐ interacting protein) (E3 ubiquitin protein ligase CHIP) (CLL‐ associated antigen KW‐8) (Antigen NY‐CO‐7).

binding; ubiquitin‐protein ligase activity; proteasomal ubiquitin‐dependent protein cat; protein folding; protein maturation; protein polyubiquitination;

F ‐ Protein Fate 6 0.229 0.062 2.187 0

ubiquitin‐dependent SMAD protein catabolism

PIN4 Q9Y237

PIN4 Peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 4 (EC 5.2.1.8) (Rotamase Pin4) (PPIase Pin4) (Parvulin 14) (Par14) (Peptidyl‐prolyl cis/trans isomerase EPVH)

mitochondrial matrix; protein folding

F ‐ Protein Fate 2 ‐0.067 2.093 2.187 0

(hPar14).

MAST1 Q9Y2H9

MAST1 Microtubule‐associated serine/threonine‐protein kinase 1 ( ) (

ATP binding; magnesium ion binding; protein serine/threonine kinase activity; cytoskeleton

dC ‐ Cellular Fate and

44 N/A 0 3.156 3.732MAST1 Q9Y2H9(EC 2.7.11.1) (Syntrophin‐associated serine/threonine‐protein kinase).

organization and biogenesis; protein amino acid phosphorylation; protein kinase cascade

Organization44 N/A 0 3.156 3.732

MOB3 Preimplantation protein 3 (Mps one binder kinase activator

PREI3 Q9Y3A3(Mps one binder kinase activator‐like 3) (Mob1 homolog 3) (Mob3) (Class II mMOB1) (2C4D).


Note: Red‐highlighted cells contain statistically significant values (see Materials and Methods)

ANTXR1 Q9H6X2CENTD2 Q96P48

Protein

NameSPID

Supplementary Table S2. A list of the 133 EGFR‐interacting proteins identified by Blagoev et al. (2) and Jones et al. (3). Only the protein name and SPID are provided.

CENTD2 Q96P48GIT2 Q14161TLN1 Q5TCU5ARHGEF7 Q14155LIMD1 Q9UGP4AP2B1 P63010CST3 P01034PIK3R3 Q92569Adapter O00428NME2 P22392DSG2 Q14126RALY Q9UKM9RALY Q9UKM9ITLN2 Q8WWU7ANXA2 P07355SRI P30626SLA2 Q9H6Q3TFRC P02786HNRPR O43390IQGAP1 P46940ACTR2 P61160CDNA: Q9H706ACTR3 P61158CDH2 P19022CDH2 P19022Gamma‐ Q86W21DAB2 P98082CDNA Q6ZVM7EFHD2 Q96C19MAPK14 Q16539SGK223 Q86YV5MVP Q14764AXL P30530ARPC4 P59998PDCD6IP Q6NUS1ARPC3 O15145ARPC3 O15145WBP2 Q969T9ARPC2 O15144ARPC1B O15143

STAM Q92783RIN1 Q13671VIM P08670CCDC50 Q8IVM0APBB2 Q5I0G1HNRPA2B1 P22626HNRPA2B1 P22626HECTD1 Q9ULT8CDNA Q9H7Z5DDX3X O00571GIT1 Q9Y2X7RPS27A Q5RKT7SDCBP O00560TOM1L1 O75674SHC1 Q5T183TNS4 Q6PJP3DKFZp686J Q5H9R6CTG3a Q9Y4I7CTG3a Q9Y4I7ACP1 P24666PRKX P51817AIDA1C Q5XLJ0hnRNP Q7KZ75Cytokine‐ Q71V34TUBB2C P68371ndp52 Q13137CYLD Q9NQC7DOK1 Q99704

HSPD1 P10809

Protein

NameSPID

Supplementary Table S3. A list of the 261 EGFR‐interacting proteins found in I2D. Data are from version 1.71 of I2D (http://ophid.utoronto.ca/i2d). Onlythe protein name and SPID are provided.

HSPD1 P10809DKFZp434 Q9UFY1LRRC17 Q8N6Y2CAMLG P49069CTNND1 O60716AMH P03971KRT6A P02538VIL2 P15311PRKCD Q05655KRT14 P02533GAB1 Q13480PIK3R3 Q5T4P2PIK3R3 Q5T4P2CAV1 Q03135LRRC59 Q96AG4PIK3C2A O00443TUBB P07437PDCD6IP Q8WUM4IRS4 O14654CTNNB1 P35222CTNNA1 P35221VAV1 Q96D37ACTA2 P62736GAB2 Q9UQC2GAB2 Q9UQC2HSPA5 P11021SLC25A3 Q00325CDC25A P30304CASP1 P29466TUBA3 Q71U36TRIM29 Q14134INPP5B P32019PXN P49023TNS4 Q8IZW8PTPN1 P18031SNRPD2 P62316SNRPD2 P62316DNAJA1 P31689CDH1 P12830EFNB1 P98172

Transformi Q9UKM3CMTM8 Q8IZV2AR P10275PLD1 Q13393PLEC1 Q15149CAV2 P51636CAV2 P51636PIK3C2B O00750HNRPK P61978PKP2 Q99959S100A9 P06702YWHAE P62258TBC1D16 Q8TBP0MAP3K14 Q99558PRKD1 Q15139MAP2K1 Q02750CD82 P27701TLR2 O60603TLR2 O60603EIF2B3 Q9NR50CSE1L P55060CAV3 P56539CSK P41240TBRG4 Q969Z0DNAJA3 Q96EY1MAP4K1 Q92918CEBPB P17676GNAI2 P04899CDCP1 Q9H5V8PRKAR1A P10644PRKAR1A P10644APBB2 Q92870PTPN2 P17706EREG O14944EPHB4 P54760ADRBK1 P25098MUC1 P15941RPS27A P62988NRG1 Q02297PLD2 O14939CLTC Q00610CISH Q9NSE2CISH Q9NSE2BTC P35070ATP5C1 P36542AGTR1 P30556

ATP2A2 P16615CLTCL1 P53675PRKCA P17252ALCAM Q13740HNRPF P52597XPO6 Q96QU8XPO6 Q96QU8PITPNA Q00169mist Q7Z7G1SHC2 P98077ERRFI1 Q9UJM3PLSCR1 O15162DNAJA2 O60884HSPA9B P38646HBEGF Q99075PKIA P61925SH2D1A O60880TLN1 Q9Y490TLN1 Q9Y490RABGEF1 Q9UJ41PTPRS Q13332TNC P24821ATP5B P06576KRT7 P08729CRKL P46109CRK P46108AREG P15514GRB10 Q13322ESR1 P03372ANKS1B Q8TAP3ANKS1B Q8TAP3FAS P25445PRKACA P17612EPHA2 P29317RGS16 O15492PEX19 P40855CBLC Q9ULV8S100A7 P31151HSPB1 P04792S100A11 P31949SFN P31947TJP1 Q07157TJP1 Q07157HNRPH1 P31943SH2B Q9NRF3DCN P07585

PTK2 Q05397SCAMP1 O15126DEGS1 O15121SEC13L1 P55735EPB41 P11171SNX6 Q9UNH7SNX6 Q9UNH7CEACAM1 P13688CDC2 P06493ITGA5 P08648PTK2B Q14289APBB1 O00213SCAMP3 O14828EEF1A1 P68104VAV2 Q5T1R9CD44 P16070KRT17 Q04695EHF Q9NZC4EHF Q9NZC4SLA Q13239TLR4 O00206SNX1 Q13596SLC3A2 P08195ZNF259 O75312SHC1 Q5T186RUTBC1 Q8IXU4LYN P07948EPPK1 P58107YES1 P07947HSPA8 P11142HSPA8 P11142MET P08581KRT8 P05787KRT18 P05783ATP1B1 P05026ATP1A1 P05023XPOT O43592KRT5 P13647CKAP4 Q07065HIST1H3A P68431ERBB3 P21860SNX4 O95219SNX4 O95219XRCC6 P12956TGFA P01135EGF P01133

SNX2 O60749PTPRJ Q12913ICAM1 P05362SH2D2A Q9NP31CAMK2G Q13555YWHAZ P63104YWHAZ P63104HD P42858FHL2 Q14192PAK1 Q13153ERBB4 Q15303HOXC10 Q9NYD6RIPK1 Q13546ATXN10 Q9UBB4HSP90AA1 P07900Tubulin P68363KPNB1 Q14974PDGFRB P09619PDGFRB P09619NUMBL Q9Y6R0HIST1H2BC P62807ARF4 P18085HIST1H1E P10412DKFZp686 Q6N089KRT1 P04264

CC BP MF

cornified envelope; cytoplasm; calcium ion

Protein

NameSPID Annotation Ontology Terms

Co-localization

TermOntology Category

Sig.

Domain

Pairs

Gene Co-

expressio

n

Semantic Similarity

ANXA1 P04083

ANXA1 Annexin A1 (Annexin I) (Lipocortin I) (Calpactin II) (Ch bi di 9) ( 35)

cytoplasm; calcium ion binding; phospholipase inhibitor activity; phospholipid binding; protein binding, bridging; receptor binding; structural molecule activity; anti‐ C ‐ Cellular Fate and N/A 0 198 0 5 183 2 013 GO 0005737ANXA1 P04083 (Chromobindin‐9) (p35)

(Phospholipase A2 inhibitory protein).

molecule activity; anti‐apoptosis; cell motility; cell surface receptor linked signal transdu; inflammatory response; keratinocyte differentiation; lipid

C Cellular Fate and Organization

N/A 0.198 0 5.183 2.013 GO:0005737

differentiation; lipid metabolism; peptide cross‐linking


Supplementary Table S4. The single EGFR‐interacting protein common to the MYTH screen, I2D, and Jones et al. and Blagoev et al. datasets.Annotation definitions are the same as those for table S1.

Protein Name SPID

PIK3R2 O00459ANKS1A Q92625IRS1 P35568

Supplementary Table S5. The 66 EGFR‐interacting proteins common to the Jones et al, Blagoev et al, and I2D datasets. Only the protein name and SPID are provided.

IRS1 P35568DOK6 Q6PKX4STAM2 O75886DOK4 Q8TEW6BCAR1 P56945MAPK8IP2 Q13387SYK P43405ABL2 P42684FER P16591DOK2 O60496PIK3R1 P27986APBA3 O96018APBA3 O96018SH2B3 Q9UQQ2EPS15 P42566APBB3 O95704SH2D3A Q9BRG2BLK P51451TNK2 Q07912NCK2 O43639EPS8 Q12929BMX P51813TUBA1 P68366MAPK8IP1 Q9UQF2MAPK8IP1 Q9UQF2RIN2 Q8WYP3INPPL1 O15357PTK6 Q13882JAK2 O60674STAT5B P51692NCK1 P16333ERBB2 P04626CLTA P09496DOK5 Q9P104VAV2 P52735NUMB P49757NUMB P49757ZAP70 P43403HGS O14964SHC3 Q92529

GRB7 Q14451FES P07332GRB14 Q14449GRB2 P62993SOCS3 O14543SHC1 P29353SHC1 P29353PTPN6 P29350CBL P22681VAV1 P15498APPL1 Q9UKG1SLC25A5 P05141BCAR3 O75815STAT5A P42229STAT1 P42224RASA1 P20936SOCS1 O15524PLCG2 P16885PLCG2 P16885SOS2 Q07890SOS1 Q07889CBLB Q13191ABL1 P00519STAT3 P40763VAV3 Q9UKW4MAPK1 P28482PLCG1 P19174SRC P12931PTPN11 Q06124

CC BP MF

cytoplasm; endoplasmic reticulum; mitochondrion;

Gene Co-

expressio

n

Semantic SimilarityProtein Name

SPID Annotation Ontology Terms Ontology CategorySig.

Domain

Pairs

Co-localization

Term

Supplementary Table S6. The 8 EGFR‐interacting protein common to the MYTH and the I2D datasets. Annotation definitions are the same those for table S1.

HSPA1A P08107HSP71 Heat shock 70 kDa protein 1 (HSP70.1) (HSP70‐1/HSP70‐2).

reticulum; mitochondrion; nucleus; unfolded protein binding; anti‐apoptosis; mRNA catabolism; response to unfolded protein

T ‐ Transcription 6 0.064 1.519 2.276 0 GO:0005737

h (YWHAQ P27348

1433T 14‐3‐3 protein theta (14‐3‐3 protein tau) (14‐3‐3 protein T‐cell) (HS1 protein).

protein binding N ‐ Not Matched 8 0.027 0 0 0.677

SH3BGRL3 Q9H299SH3L3 SH3 domain‐binding glutamic acid‐rich‐like protein 3 (SH3 domain‐ bi di i 1) (SH3BP 1)

U ‐ Uncharacterized N/A ‐0.067 0 0 0binding protein 1) (SH3BP‐1).

TXN P10599THIO Thioredoxin (Trx) (ATL‐derived factor) (ADF) (Surface‐associated

protein binding; thiol‐disulfide exchange intermediate activity; cell motility; cell proliferation;


N/A 0.148 0 5.183 1.232sulphydryl protein) (SASP).

motility; cell proliferation; cell‐cell signaling; signal transduction

Organization

cytoplasm; plasma membrane; calcium ion binding; N‐terminal

CALM1 P62158 CALM Calmodulin (CaM).binding; N‐terminal myristoylation domain binding; protein binding; G‐protein coupled receptor protein signalin

N ‐ Not Matched 16 ‐0.178 0.629 2.968 0.677 GO:0005886

SH3BGRL O75368SH3L1 SH3 domain‐binding glutamic acid‐rich‐like protein.

cytoplasm; nucleus; SH3/SH2 adaptor activity

N ‐ Not Matched N/A ‐0.208 0 0 2.272 GO:0005737acid rich like protein. SH3/SH2 adaptor activity

GAPDH P04406G3P Glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.12) (GAPDH).

cytoplasm; glyceraldehyde‐3‐phosphate dehydrogenase (p; glycolysis

E ‐ Energy Production N/A 0.034 1.090 1.456 1.232 GO:0005737

HSP90AB1 P08238HS90B Heat shock protein HSP 90‐beta (HSP 84) (HSP 90).

cytoplasm; nitric‐oxide synthase regulator activity; TPR domain binding; unfolded protein binding; positive regulation of nitric oxide biosynt; response to

N ‐ Not Matched 9 ‐0.053 1.090 8.974 2.013 GO:0005737

oxide biosynt; response to unfolded protein


CC BP MF

Co-localization

Term

Protein

NameSPID Annotation Ontology Terms Ontology Category

Sig.

Domain

Pairs

Gene Co-

expressio

n

Semantic Similarity

Supplementary Table S7. The 2 EGFR‐interacting proteins common to the MYTH dataset and the datasets of Jones et al. and Blagoev et al. Annotation definitions are the same as those for table S1.

ARPC5 O15511ARPC5 Actin‐related protein 2/3 complex subunit 5; Arp2/3 complex 16 kDa subunit; p16‐ARC

Arp2/3 protein complex; cytoplasm; protein binding; structural constituent of cytoskeleton; actin cytoskeleton organization and biogenesis; cell motility


N/A ‐0.008 1.144 5.085 0.598 GO:0005737

and biogenesis; cell motility

GSN P06396GELS Gelsolin precursor (Actin‐depolymerizing factor) (ADF) (Brevin) (AGEL)

actin cytoskeleton; cytosol; extracellular region; calcium ion binding; protein binding; actin filament polymerization; actin

N ‐ Not Matched N/A 0.097 2.093 5.079 0.677 GO:0005737(Brevin) (AGEL). polymerization; actin

filament severing; barbed‐end actin filament capping


Movie S1. Maximum projection of GFP-Rab5 endosome dynamics in mCherry-tubulin-expressing cells.

Image sequences (4 images per Z stack, 2 stacks/second) with observation time of 5 minutes and playing at

25 frames/second. Individual endosomes were identified and tracked with MotionTracker® software.

Supplementary References

1. Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M. A proteomics strategy to elucidate

functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21, 315-318 (2003).

2. Jones RB, Gordus A, Krall JA, MacBeath G. A quantitative protein interaction network for the ErbB

receptors using protein microarrays. Nature 439, 168-174 (2006).

supplementary materials for - science signaling · 2009. 12. 17. · fig. s1. generation and...

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