Supplementary Materials for - Linding Lab...Rich1 and Tuba localize to cell-cell junctions, associate with proteins involved in junction formation and cell polarity, and are active

www.sciencesignaling.org/cgi/content/full/2/98/ra76/DC1

Supplementary Materials for

Eukaryotic Protein Domains as Functional Units of Cellular Evolution

Jing Jin,* Xueying Xie, Chen Chen, Jin Gyoon Park, Chris Stark, D. Andrew James, Marina Olhovsky, Rune Linding, Yongyi Mao,* Tony Pawson*

*To whom correspondence should be addressed. E-mail: [email protected] (T.P.);

[email protected] (Y.M.); [email protected] (J.J.)

Published 24 November 2009, Sci. Signal. 2, ra76 (2009) DOI: 10.1126/scisignal.2000546

This PDF file includes:

Section S-I. Characterization of the functional relationships between RhoGEF/RhoGAP pairs Tuba/Rich1, and DEPDC1/P-Rex1. Section S-II. Correlation between protein kinase regulatory and catalytic domains. Section S-III. Web site for domain clubs across seven genomes. Section S-IV. Supplementary methods. Section S-V. Supplementary references. Table S1. Domains found in common versus domains that are unique to SH2 domain proteins or SH3 domain proteins. Fig. S1. Functional characterization of RhoGEF and RhoGAP protein pairs. Fig. S2. Domain profiling of human protein kinases (PTK) and protein tyrosine phosphatases (PTP). Fig. S3. Domain profiling of the human kinome. Fig. S4. Domain profiling across six eukaryotic kinomes. Fig. S5. Domain club cut-off selection. Fig. S6. Test of the robustness of the domain profiling method for applications in large data sets. Fig. S7. A Web site for domain club analysis—example of Dicer1 and its DSRM domain. Fig. S8. General procedure for spreading-on-graph (SOG) clustering in the analysis of the molecular environments of domain families. Fig. S9. The interactomes of the 70 domain families employed in the analysis of domain-based functional compartments. Fig. S10. An overview of the Royal Family of domains. Fig. S11. Sequence characteristics of the mouse Piwi clade of the Argonaute proteins. Fig. S12. An MS/MS spectrum of a tryptic peptide containing dimethylated (2me) Arg53 of endogenous Miwi protein from testis.

Supplementary - Eukaryotic protein domains as functional units of cellular

evolution.

Jing Jin, Xueying Xie, Chen Chen, Jin Gyoon Park, Chris Stark, D. Andrew James,

Marina Olhovsky, Rune Linding, Yongyi Mao and Tony Pawson

Section S-I. Characterization of the functional relationships between

RhoGEF/RhoGAP pairs Tuba/Rich1, and DEPDC1/P-Rex1.

Human proteins with RhoGEF and RhoGAP domains were clustered based on their

non-catalytic interaction domains (Fig. 2 and fig. S1A). We have explored whether co-

clustered GEFs and GAPs may have related functions by focusing on two clusters that

share a BAR domain or a DEP domain, respectively (Fig. 2, boxes B and C respectively).

BAR domains, present in GEFs (Rich1, SH3BP1 and KIAA0672) and GAPs

(Tuba/Dnmbp, FLJ41603 and ENSG00000188436) in Cluster B (blue box in fig. S1A),

can potentially bind phospholipids and curved membranes (1). Rich1 and related proteins

have an N-terminal BAR domain, a central GAP domain, and a C-terminal proline-rich

sequence with SH3 domain-binding sites, whereas Tuba has a BAR domain immediately

following the RhoGEF domain (fig. S1B). Tuba also has four N-terminal SH3 domains

which selectively bind dynamin, a GTPase involved in endocytosis, and two C-terminal

SH3 domains (fig. S1B), previously shown to recruit actin regulatory proteins, including

N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1 and Ena/VASP proteins (2). Both

Rich1 and Tuba localize to cell-cell junctions, associate with proteins involved in

junction formation and cell polarity, and are active against the Cdc42 GTPase (3, 4). In

an analysis of binding partners for the C-terminal SH3 domains of Tuba, we identified a

S-1

number of known and novel targets involved in processes such as the formation of cell

junctions, cytoskeletal regulation and vesicle trafficking (fig. S1B). Some of these

proteins, including MUPP1, CD2AP and CIN85 also associate with Rich1 (fig. S1B,

asterisks) (4), directly or indirectly. These data suggest that Tuba and Rich1 impinge on

common aspects of cellular function, consistent with the notion that co-clustering based

on domain composition is indicative of a functional relationship.

Proteins in cluster C have GEF (P-Rex1 and DEPDC2/P-Rex2) or GAP (DEPDC1

and DEPDC1b) domains, in conjunction with a DEP domain (fig. S1C), which has been

variously implicated in membrane association and interactions with G protein coupled

receptors (5, 6). Of these, P-Rex1 has a Rac-specific GEF activity that is stimulated by

PI(3,4,5)P3 and Gβγ signalling (7). The GAP domain of DEPDC1 lacks an arginine

residue that is normally critical for GAP activity (not shown), and its precise role in

regulating Rho GTPases remains uncertain. It reportedly has a nuclear location (8), which

we have confirmed by transfection of Flag-tagged DEPDC1 into HEK293 cells (fig. S1C,

right), a finding that appears contrary to the involvement of other DEP domain proteins

with signalling at the plasma membrane. However, upon breakdown of the nuclear

envelope during mitosis, DEPDC1 becomes localized to the cell cortex (fig. S1C, right).

In addition, over-expression of P-Rex1, which is cytoplasmic, led to the relocalization of

DEPDC1 to the plasma membrane as judged by immunofluorescence, even in interphase

cells (fig. S1C, right). A similar recruitment of DEPDC1 from the nucleus to the

cytoplasm upon P-Rex1 over-expression was seen following cell fractionation (fig. S1C,

bottom). Over-expression of a distinct Rac-specific GEF, Tiam1, did not influence

DEPDC1 localization (data not shown). These results are consistent with the hypothesis

S-2

that ancillary interaction domains may target GEF and GAP proteins to related cellular

processes, although it does not necessarily imply that such GEF/GAP pairs work directly

in tandem.

Section S-II. Correlation between protein kinase regulatory and catalytic domains

The human genome encodes 518 protein kinases, which fall into ten classes based

on the sequence relationships and functional properties of their catalytic domains (9).

About half of these protein kinases also possess one or more interaction domains. These

are a common feature of some sub-groups, such as TK, TKL and RGC kinase families

(fig. S3, top panel), and can have diverse functions, including regulating catalytic

activity, targeting the kinase to specific subcellular locations, and interacting with

activators and substrates. Some kinases (e.g CK and CMGC family members) do not

possess intrinsic non-catalytic domains (fig. S3, top panel), but are often associated with

regulatory domain-based subunits; we have not considered such regulatory proteins in the

analysis outlined below.

We used domain profiling to explore whether multi-domain kinases can be grouped

based on their non-catalytic domains, and whether the resulting clusters are related to the

grouping obtained by sequence analysis of kinase domains. To this end, we analyzed all

annotated human protein kinases by hierarchical clustering, based on their domain

composition after removal of the common kinase domain (fig. S3, bottom panel). The

resulting matrix, with domains arrayed on the x-axis and kinases on the y-axis, reveals

groups of kinases that cluster because they possess related non-kinase domains (detailed

in panels a-f). Kinases are color-coded by their position on the kinome tree, which is

S-3

largely based on the sequences of their kinase domains. This analysis reveals a selective

relationship between kinase and interaction domains. For example, the tyrosine kinase

(TK) group largely separates away from serine/threonione kinases, and forms two

clusters (d and e) that contain receptor tyrosine kinases (RTK) on the one hand (cluster d)

and cytoplasmic tyrosine kinases on the other (cluster e). The RTKs are co-clustered due

to the presence of domains such as Ig, FN3 and SAM, found in both their extracellular

and cytoplasmic regions, whereas the cytoplasmic tyrosine kinases are grouped through

the frequent presence of SH2, SH3, PH, Btk and B41/FERM domains. In the case of

serine/threonine kinases, the largest cluster (cluster c) is primarily formed by members of

the AGC family, which are mostly basophilic kinases that are co-clustered on the matrix

because they contain domains such as C1, C2, PH, PB1 and RGS.

We also conducted a co-clustering analysis using the non-catalytic domains of

human protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) groups.

We observed co-clusters of PTKs and PTPs due to the presence of common SH2 and

FERM domains (fig. S2). Similarly, receptor tyrosine kinases (RTKs) and

transmembrane PTPs co-cluster due to common domains such as FN3 and Ig family

members (fig. S2). It is less common for serine/threonine kinases and phosphatases to

share common domains (data not shown), potentially because both classes of enzymes

employ a diverse array of regulatory subunits (10).

Furthermore, we used domain profiling to cluster protein kinases from

Saccharomyces cerevisiae (Sc), Dictyostelium discoideum (Dd), Caenorhabditis elegans

(Ce), Drosophila melanogaster (Dm), Mus musculus (Mn) and Homo sapiens (Hs), based

on their non-catalytic domains (fig. S4, left panel). This shows the extent to which

S-4

kinases from different species contribute to the various domain-based clusters (detailed in

boxes a-i). As examples, clusters a and b (equivalent to clusters d and e in S3) primarily

contain tyrosine kinases, which show marked expansions and contractions between

species. For example, receptor tyrosine kinases, most notably the Eph receptor TK group

with SAM and FN3 domains have markedly increased from invertebrates to vertebrates

(box a). Cytoplasmic TKs appear in cluster b, which shows the presence of SH2-

containing kinases (Shk1-5) in Dictyostelium, each with an SH2 domain linked to a TKL

catalytic domain, a striking expansion of C. elegans kinases with SH2 and TK domains

(11), and an increase in the numbers of SH2-containing kinases with additional

interaction domains, such as SH3, in vertebrates. In contrast, yeast (Sc) and Dictyostelium

(Dd), both lacking a bona fide tyrosine kinase, are only represented in box a by Ste11

family kinases with a SAM domain linked to a serine/threonine kinase (STK) domain.

The results indicate that several families of protein kinases can be similarly grouped

either by the sequence relationships of their kinase domains, or by unbiased hierarchical

clustering of their non-catalytic interaction domains. The results support a model in

which the catalytic and interaction domains of protein kinases have co-evolved to allow

appropriate physiological regulation of kinase activity and selection of specific

substrates.

Section S-III. Website for domain clubs across seven genomes – Readers can display

any individual domain file (domain terms defined in SMART and pfam) from an

interactive website http://pawsonlab.mshri.on.ca/DomainClub/domainClub.php. The

website allows the reader to search by protein and domain names. An analysis of protein

S-5

http://pawsonlab.mshri.on.ca/DomainClub/domainClub.php

Dicer and an evolutionary overview of its DSRM domain are presented as an example

(also in fig. S7).

S-6

Section S-IV. Supplementary methods

Experimental procedures for transfection, affinity chromatography, mass

spectrometry, immunofluorescence and nuclear fractionation

Miwi, mAgo1 and Tdrd1 cDNAs were derived from MGC:150072, 150309 and

72119 respectively. Miwi and mAgo1 protein-coding sequences are fused in-frame to an

N-terminus FLAG sequence in a pcDNA3 vector. Tdrd1 is expressed using a Creator

EGFP vector system (12). FLAG-M2 antibody was from Sigma-Aldrich; EGFP antibody

was from Abcam (ab290). For endogenous Miwi immunoprecipitation, approximately 0.4

gram of 4-week mouse testis tissue was homogenized and lysed in buffer containing

0.5% NP40, 10 mM HEPES pH 7.5, 150 mM NaCl2, 1 mM EDTA, and 2 μg of anti

Hiwi/Miwi antibody (from Abcam) was used. The immunoprcipitated Miwi protein was

separated by SDS-polyacrylamide gel electrophoresis, isolated from a gel band, and

subjected to standard proteolytic digestion using trypsin. Resulting peptides were

analyzed in the QSTAR Elite Hybrid LC/MS/MS System (from the Applied

Biosystems/MDS Sciex). Peptide identification (including modifications) was assisted by

mass spectrometry software Protein Pilot (from BC Proteomic Network). Transfection of

HEK293T cells and co-immunoprecipitation assays have been described previously (13).

Full length human cDNA for Tuba/Dnmbp was derived from clone

DKFZp451J178. Recombinant GST fusion proteins containing Tuba SH3 domains 1-4

(residues 1-315) and SH3 domains 5-6 (residues 1276-1577) were expressed and purified

following the standard pGEX-4t-2 protocol (GE Healthcare Life Sciences). MYC-epitope

S-7

tagged P-Rex1 expression vector was a gift from Dr. Heidi Welch. Full-length human

DEPDC1 coding cDNA sequence was derived from MGC:70715, and then fused in-

frame to an N-terminus FLAG sequence in a pcDNA3 vector. Myc-9E10, Histone H1

(FL-219), Dynamin-H300, Mena-H50, WASP-H100, CIN85-H300 and CD2AP-B4

antibodies were from Santa Cruz; Zo-1 and MUPP1 antibodies were from Zymed.

General methods for protein identification from affinity chromatography using LC-

tandem mass spectrometry and methods for transfection and immunofluorescence

microscopy were described previously (13). Nuclear-cytoplasmic fractionation

experiment was carried out following a modified protocol from abcam.com. Briefly, one

day after transfection, HEK293 cells were lysed on ice in buffer containing 0.05% NP40,

10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT. Supernatant

representing the cytoplasmic fraction was collected from low-speed centrifugation. To

extract a nuclear fraction, the pellet was resuspended in buffer containing 300 mM NaCl,

5 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol

(v/v).

Section S-V. Supplementary references

1. B. J. Peter, H. M. Kent, I. G. Mills, Y. Vallis, P. J. Butler, P. R. Evans et al., BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495-499 (2004).

2. M. A. Salazar, A. V. Kwiatkowski, L. Pellegrini, G. Cestra, M. H. Butler, K. L. Rossman et al., Tuba, a novel protein containing bin/amphiphysin/Rvs and Dbl homology domains, links dynamin to regulation of the actin cytoskeleton. J Biol Chem 278, 49031-49043 (2003).

3. T. Otani, T. Ichii, S. Aono, M. Takeichi, Cdc42 GEF Tuba regulates the junctional configuration of simple epithelial cells. J Cell Biol 175, 135-146 (2006).

S-8

4. C. D. Wells, J. P. Fawcett, A. Traweger, Y. Yamanaka, M. Goudreault, K. Elder et al., A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. Cell 125, 535-548 (2006).

5. J. D. Axelrod, J. R. Miller, J. M. Shulman, R. T. Moon, N. Perrimon, Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 12, 2610-2622 (1998).

6. M. Boutros, N. Paricio, D. I. Strutt, M. Mlodzik, Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109-118 (1998).

7. H. C. Welch, W. J. Coadwell, C. D. Ellson, G. J. Ferguson, S. R. Andrews, H. Erdjument-Bromage et al., P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac. Cell 108, 809-821 (2002).

8. M. Kanehira, Y. Harada, R. Takata, T. Shuin, T. Miki, T. Fujioka et al., Involvement of upregulation of DEPDC1 (DEP domain containing 1) in bladder carcinogenesis. Oncogene 26, 6448-6455 (2007).

9. G. Manning, D. B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome. Science 298, 1912-1934 (2002).

10. D. M. Virshup, S. Shenolikar, From promiscuity to precision: protein phosphatases get a makeover. Mol Cell 33, 537-545 (2009).

11. G. D. Plowman, S. Sudarsanam, J. Bingham, D. Whyte, T. Hunter, The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc Natl Acad Sci U S A 96, 13603-13610 (1999).

12. K. Colwill, C. D. Wells, K. Elder, M. Goudreault, K. Hersi, S. Kulkarni et al., Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites. BMC Biotechnol 6, 13 (2006).

13. J. Jin, F. D. Smith, C. Stark, C. D. Wells, J. P. Fawcett, S. Kulkarni et al., Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr Biol 14, 1436-1450 (2004).

S-9

table S1. Domains found in common (asterisks and color) versus domains that are unique to the human SH2 domain proteins or SH3 domain proteins.

supplementary Table S1 -- Jin et al.

SH2 SH3

B 41/FE R M * B 41/FE R M *B T K * B T K *C 1* C 1*C 2* C 2*C H * C H *FA B D * FA B D *FC H * FC H *P H * P H *P LC X c* P LC X c*P LC Yc* P LC Yc*P T B * P T B *R A * R A *R asG A P * R asG A P *R hoG A P * R hoG A P *R hoG E F* R hoG E F*R IN G * R IN G *S A M * S A M *S H 2* S H 2*S H 3* S H 3*S T YK c* S T YK c*T yrK c* T yrK c*U B A * U B A *IP P c A D FP T P c A N KP T P c_D S P c A rfG apR asG E F B A RS 1 B P 1S O C S E fhV P S 9 FN 3YqgFc G uK c

IGIQL27M YS cM yT H 4N E BP D ZP XR U NS _T K cS E C 14S orbS P E CT B CT P RU IMV H SW D 40W WZU 5

KIAA0672ARHGAP17/RICH1SH3BP1DNMBP/TUBAFLJ41603ENSG00000188436

low

_com

pl.

Rho

Gap

Rho

Gef

PH BAR

coile

d_co

ilFC

HSH

3

A B

C

domain

protein

low

_c

om

ple

xit

yR

ho

Ga

pR

ho

Ge

fP

H

STA

RT

SA

M_

2B

AR

co

ile

d_

co

il_

reg

ion

FC

HS

H3

C1

SH

2C

HC

2E

Fh

EH

HD

AC

_in

tera

ct

FF

SP

EC

SE

C1

4IG

c2

S_

TK

cF

N3

IG_

lik

eB

41

PH

FY

VE

SA

MA

rfG

ap

RA

MY

Sc

IQ His

ton

eR

asG

EF

CR

asG

EF

ND

EP

PD

ZR

BD

RG

SV

PS

9M

OR

NW

WM

yth

4sig

na

l_p

ep

tid

eL

RR

FO

LN

EG

F_

lik

eL

RR

NT

LR

R_

TY

PL

RR

CT

La

mG

CT

EG

F_

CA

IPP

cB

RC

T

GAP GEF

ENSG00000114346 ECT2ENSG00000204084 INPP5BENSG00000122126 OCRLENSG00000147256 RP13-102H20.1ENSG00000124143 C20orf95ENSG00000187122 ARHGAP19ENSG00000164741 DLC1ENSG00000130052 STARD8ENSG00000133121 STARD13ENSG00000156299 TIAM1ENSG00000146426 TIAM2ENSG00000107863 ARHGAP21ENSG00000196914 ARHGEF12ENSG00000132694 ARHGEF11ENSG00000099331 MYO9BENSG00000066933 MYO9AENSG00000089639 GMIPENSG00000137962 ARHGAP29ENSG00000161800 RACGAP1ENSG00000116584 ARHGEF2ENSG00000170776 AKAP13ENSG00000180448 HMHA1ENSG00000097096 SYDE2ENSG00000017797 RALBP1ENSG00000171680 PLEKHG5ENSG00000138640 FAM13A1ENSG00000031003 C5orf5ENSG00000104880 ARHGEF18ENSG00000003393 ALS2ENSG00000106069 CHN2ENSG00000128656 CHN1ENSG00000164341 RGNEFENSG00000047365 CENTD1ENSG00000186635 STARD10ENSG00000120318 CENTD3ENSG00000137727 ARHGAP20ENSG00000167433 ARHGAP23ENSG00000075884 ARHGAP15ENSG00000152767 FARP1ENSG00000006607 FARP2ENSG00000127084 FGD3ENSG00000139132 FGD4ENSG00000146192 FGD2ENSG00000180263 FGD6ENSG00000154783 FGD5ENSG00000102302 FGD1ENSG00000071205 ARHGAP10ENSG00000138639 ARHGAP24ENSG00000079482 OPHN1ENSG00000163219 ARHGAP25ENSG00000128805 ARHGAP22ENSG00000186716 BCRENSG00000159842 ABRENSG00000185602 ARHGAP27ENSG00000165322 ARHGAP12ENSG00000115904 SOS1ENSG00000100485 SOS2ENSG00000058335 RASGRF1ENSG00000113319 RASGRF2ENSG00000126217 MCF2LENSG00000053524 MCF2L2ENSG00000101977 MCF2ENSG00000186654 ARHGAP8ENSG00000175220 ARHGAP1ENSG00000160145 KALRNENSG00000038382 TRIOENSG00000006740 KIAA0672ENSG00000140750 ARHGAP17/RICH1ENSG00000100092 SH3BP1ENSG00000107554 DNMBP/TUBAENSG00000183111 FLJ41603ENSG00000188436ENSG00000198399 ITSN2ENSG00000205726 ITSN1ENSG00000196935 SRGAP1ENSG00000089820 ARHGAP4ENSG00000196220 SRGAP3ENSG00000163486 SRGAP2ENSG00000131089 ARHGEF9ENSG00000066248 NGEFENSG00000050327 ARHGEF5ENSG00000136002 ARHGEF4ENSG00000182957 SPATA13ENSG00000142632 ARHGEF19ENSG00000004777 SNX26ENSG00000130762 ARHGEF16ENSG00000114790 SGEF(HMFN1864, DKFZp434D146)ENSG00000145819 ARHGAP26ENSG00000123329 ARHGAP9ENSG00000129675 ARHGEF6ENSG00000102606 ARHGEF7ENSG00000141968 VAV1ENSG00000160293 VAV2ENSG00000134215 VAV3ENSG00000145675 PIK3R1ENSG00000105647 PIK3R2ENSG00000154358 OBSCNENSG00000046889 DEPDC2ENSG00000124126 PREX1ENSG00000024526 DEPDC1ENSG00000035499 DEPDC1BENSG00000147799 KIAA1688ENSG00000160007 GRLF1ENSG00000100852 ARHGAP5

ENSG00000198826 ARHGAP11AENSG00000164691 TAGAPENSG00000134909 DKFZp451F1115(grit, RICS)ENSG00000047648 ARHGAP6ENSG00000169662 NP_997357.1ENSG00000105137 SYDE1ENSG00000169668 NP_997357.1ENSG00000187951 FAM7A2ENSG00000186517 ARHGAP30ENSG00000031081 CDGAPENSG00000165895 FLJ32810ENSG00000088756 ARHGAP28ENSG00000146376 ARHGAP18ENSG00000163947 ARHGEF3ENSG00000137135 C9orf100(RP11-331F9.7)ENSG00000104728 ARHGEF10ENSG00000196155 PLEKHG4ENSG00000198844 ARHGEF15ENSG00000153404 KIAA1909ENSG00000120278 PLEKHG1ENSG00000090924 PLEKHG2ENSG00000074964 ARHGEF10LENSG00000076928 ARHGEF1ENSG00000110237 ARHGEF17ENSG00000126822 PLEKHG3ENSG00000165801 FLJ00128(FLJ10357, FLJ00056)ENSG00000135502 SLC26A10ENSG00000187510 FLJ46688ENSG00000008323 PLEKHG6ENSG00000173848 NET1

C

B

DEPDC2PREX1DEPDC1DEPDC1B

DEP

PDZ

MYC (P-Rex1)

FLAG (DEPDC1)

C N C N C N

MYC-P-Rex1

FLAG-DEPDC1

Histone H1

DEPDC1

DEPDC1

DEPDC1

DEPDC1

P-Rex1

P-Rex1

DAPI

DAPI

DAPI

composite

composite+Actin

composite+Actin

composite

FLAG-DEPDC1+ MYC-P-Rex1

FLAG-DEPDC1

Dynamin

Zo-1

WASP

Coomassie

1 2 3 4

GS

T-S

H3

(1-4

)

GS

T-S

H3

(5,6

)

GS

T

Tota

l lys

ate

CD2AP*

MUPP1*CIN85*

Mena

Protein Mascot Score

Cell junction:Afadin 665Zo-1 396Zo-2 347Scribble 104MUPP1* 80

Cytoskeleton:Mena 415N-WASP 414 α-actinin 261Wire 145WAVE 124diaphanous 1 94NCKAP1 92KIF15 45

Vesicle trafficking:CD2AP* 513RIN3 409CIN85/SH3KBP1* 245EXOSC10 107LYST 32

Function unknown:CrkRS/cdc2L5 466ZAP3 429Bat2 266

* also interacting with Rich1 (Wells et al.)

affinity chromatographyGST-Tuba-SH3(5,6) affinity purification

supplementary Figure S1 -- Jin et al.

Proline-rich region

SH3 BARGEFSH3 SH3SH3 SH3SH3 Tuba

GAPBAR Rich1,SH3BP1,KIAA0672

WB:

++++ -

-

fig. S1. Functional characterization of RhoGEF and RhoGAP protein pairs

A. Domain profiling of human RhoGAP and RhoGEF proteins, as in Figure 2. B. A

cluster of GEF and GAP proteins containing BAR domains (blue box in A) (top panel),

and their corresponding domain architectures (middle panel); Bottom panel:

Identification of proteins that interact with SH3 domains 1-4 or 5-6 of Tuba. Affinity

chromatography using GST fusion proteins containing Tuba SH3 domains 5-6 was

conducted to purify protein from HEK-293 cell lysate, and associated proteins were

identified through mass spectrometry (left). Selected examples were confirmed by

western blotting (right - baits marked with arrows on coomassie-stained gel). C.

Relationships between P-Rex1 and DEPDC1. Top left: Co-clustering of DEP domain-

containing GEF and GAP proteins (marked with the purple box in A). Upper-right:

Subcellular localization of FLAG-DEPDC1 in HEK-293 cells (Staining with anti-FLAG

M2 antibody. Arrow: mitotic cell). Lower-right: Over-expression of P-Rex1 (MYC-

tagged) causing translocation of FLAG-DEPDC1 to the cell cortex. Bottom-left: Nuclear

(N) and cytoplasmic (C) fractionation of DEPDC1. Western blots compare the nuclear

and cytoplasmic expressions of transfected FLAG-DEPDC1 under the conditions

whereby FLAG-DEPDC1 had been co-transfected with either an empty pcDNA3 vector

(-) or MYC-P-Rex1.

PTPc

TyrK

cST

Ykc

Pfam

:CR

AL_

TRIO

_NSE

C14

Pfam

:BR

O1

LDLa

WIF

CA

FA58

CFA

BD

SH2

SH3

BTK

PH FCH

Pfam

:FA

FER

MPf

am:F

ERM

_CPD

ZK

IND

LRR_

CT

LRR_

NT

Pfam

:Rec

ep_L

_do

mai

nFU LR

RC

TPf

am:L

RR_1

LRRN

TEP

H_l

bd

SAM

FN3

Pfam

:Ig_T

ie2_

1EG

F(EG

F)EG

F_lik

eIG

_lik

eIG IG

c2M

AM

Pfam

:Car

b_a

nh

ydra

seK

RLY Pf

am:F

zSe

ma

IPT

PSI

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

LTKTYRO3psFERpsTCPTP/PTPN2IA2beta/PTPRN2PTP1B/PTPN1PEST/PTPN12STEP/PTPN5PTPTyp/PTPN20HePTP/PTPN7PTPepsilon/PTPREPTPalpha/PTPRAIA2/PTPRNPCPTP1/PTPRRLyPTP/PTPN22BDP1/PTPN18LMR2LMR3SuRTK106FLT1psJAK2_HumanLMR1

Protein Tyrosine Kinase (PTK)

Protein Tyrosine Phosphatase (PTP)

Domain

Protein


fig. S2. Domain profiling of human protein tyrosine kinases (PTK) and protein

tyrosine phosphatases (PTP)

Two-way hierarchical clustering of human PTK (red) and PTP (blue) by their non-

catalytic domains (the catalytic domains are listed in separate columns at left). Domains

such as SH2, FERM, FN3 and IGs (in bold) are present in both PTK and PTP proteins.

Proteins possessing these domains are indicated in yellow.

MIT

PX

BR

OM

OR

ING

BB

OX

PH

DB

BC

CA

RD

WD

40sm

all_

GTP

ase

Pfa

m:N

UC

194

Pfa

m:F

AT

Pfa

m:F

ATC

PI3

Kc

Pfa

m:H

EA

TU

ME

Pfa

m:R

AP

Pfa

m:F

AS

T_2

Pfa

m:F

AS

T_1

PA

SD

SR

MR

IOC

AD

CX

Pfa

m:R

CC

1M

ad3_

BU

B1_

ILD

LaW

IFTU

DO

RR

RM

Pfa

m:A

BC

1P

fam

:HS

P20

FA58

CcN

MP

Pfa

m:K

A1

UB

AP

fam

:PO

LO_b

oxP

fam

:LR

R_1

Pfa

m:Y

LPP

fam

:Rec

ep_L

_dom

ain

FU EP

H_l

bdS

AM

FN3

Pfa

m:F

zK

RIG

c2IG

_lik

eIG P

fam

:EG

F_2

EG

FE

GF_

like

Pfa

m:A

lpha

_kin

ase

Pfa

m:Io

n_tra

nsP

fam

:Sel

1IQ M

YS

cLY FC

HP

B1

C2

Hr1

Rho

GE

FS

EC

14S

PE

CR

hoG

AP

RB

DC

1P

HC

NH

PB

DS

H2

SH

3B

TKP

fam

:HR

1R

GS

Pfa

m:F

ocal

_AT

B41

L27

GuK

cP

DZ

LIM

Pfa

m:C

aMK

II_A

DR

HO

DTB

CP

SI

Sem

aIP

TP

UG

PQ

QA

NK

DE

ATH

FHA

AD

FP

fam

:RIO

1P

fam

:UB

AG

SP

fam

:Act

ivin

_rec

pD

naJ

Pfa

m:A

NF_

rece

ptor

CY

Cc

Pfa

m:tR

NA

-syn

t_2b

RW

DH

ATP

ase_

c

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

CAMK

TK

RGC

Atypical

STE

TKL

Other

AGC

BARK1BARK2RHOKGPRK4GPRK7GPRK5GPRK6PKN3PKN1PKN2KSR1KSR2PKCtPKCePKCgPKCbPKChPKCaPKCdBRAFRAF1ARAFCRIKMRCKaMRCKbPKD3DMPK2ROCK2PKD2PKD1ROCK1AKT2AKT1AKT3PKCzPKCi

BMXBTKTECITKYESABLSRMFGRARGCSKTXKBLKLCKLYNFYNSRCFRKHCKBRKCTKSYKZAP70MLK2ACKMLK4TNK1MLK3MLK1FESFERFAKPYK2TYK2JAK1JAK3JAK2

TAF1LBRDTBRD4BRD3BRD2TAF1TIF1gTIF1bTIF1aMAP3K1

ObscnTradTrioBCRCASK

HER4/ErbB4IGF1RIRRINSREGFRHER2/ErbB2HER3/ErbB3ROSMERSPEGTIE1TIE2EphA3EphB1EphA8EphA1EphB4EphA5EphA10EphB2EphA7EphA4EphA2EphB6EphB3EphA6ZAKTTNsmMLCKTYRO3AXLAlphaK1KITCCK4PDGFRbPDGFRaFLT1KDRFGFR3FMSFLT4ROR2FLT3FGFR4TRKCTRKATRKBFGFR1FGFR2ROR1MUSK

FRAPDNAPKATMTRRAPSMG1ATRPIK3R4SCYL3SCYL1FusedRSKL1

BR

OM

OR

ING

BB

OX

PH

DB

BC

WD

40sm

all_

GTP

ase

Pfa

m:N

UC

194

Pfa

m:F

AT

Pfa

m:F

ATC

PI3

Kc

Pfa

m:H

EA

TU

ME

PB

1C

2H

r1R

hoG

EF

SE

C14

SP

EC

Rho

GA

PR

BD

C1

PH

CN

HP

BD

SH

2S

H3

BTK

Pfa

m:H

R1

RG

S

Pfa

m:L

RR

_1P

fam

:YLP

Pfa

m:R

ecep

_LFU E

PH

_lbd

SA

MFN

3P

fam

:Fz

KR

IGc2

IG_l

ike

IG Pfa

m:E

GF_

2E

GF

EG

F_lik

e

PH

CN

HP

BD

SH

2S

H3

BTK

Pfa

m:H

R1

RG

SP

fam

:Foc

al_A

TFE

RM

FN3

Pfa

m:F

zK

RIG

c2IG

_lik

eIG P

fam

:EG

F_2

EG

FE

GF_

like

Pfa

m:A

lpha

_kin

ase

Pfa

m:Io

n_tra

nsP

fam

:Sel

1IQ M

YS

cLY FC

HP

B1

C2

Hr1

Rho

GE

FS

EC

14S

PE

CR

hoG

AP

RB

DC

1P

HC

NH

PB

DS

H2

SH

3

aa

b

b

c

c

d

d

e

e

f

f

domain

protein

12

30

2439

4725

55

24

26 195

85

1

3610

0

61

0

0

5TK TKL RGC STE

CAMK CK CMGCOther

AGC

Atypical

other domain beside kinase

only kinase domain


fig. S3. Domain profiling of the human kinome

Human protein kinases were each annotated for their domain composition. Each pie-chart

(upper panel) represents a distinct functional sub-group, and indicates the number of

kinases within each group that contain or lack non-kinase domains. Lower panels:

Kinases were clustered through their non-catalytic domains (left, pixel color indicating

kinase class). To the right: Magnified version of selected clusters (boxes a-f).

Pfam

:RA

PPf

am:F

AST

_2Pf

am:F

AST

_1C

ARD

HEC

TcPf

am:R

CC

1M

ORN

DC

XD

SPc

Pfam

:Co

llag

enPf

am:C

ol_

cuti

cle_

NLD

LaPf

am:M

AM

Mad

3_B

UB

1_I

Pfam

:Mad

3_B

UB

1_II

WIF

RRM

MAT

HPf

am:F

NIP

Pfam

:HG

TP_a

nti

cod

on

RW

DPf

am:tR

NA

-syn

t_2b

RHO

DTB

CC

ASh

KT

PSI

IPT

Sem

aH

15D

SRM

MA

DF

HO

XPf

am:A

BC

1Pf

am:P

OLO

_box

PQQ

PUG

Pfam

:SA

M_1

Pfam

:SA

M_2

AN

KD

EATH

BTB

Pfam

:An

kPH

DB

BC

BB

OX

RIN

GB

RO

MO

Kel

chAT

_ho

ok

Zn

F_C

2HC

cNM

PPf

am:R

asPf

am:M

iro

Ras

GEF

NR

asG

EFG

RA

MPf

am:R

asG

EF_N

Pfam

:Myo

tub

-rel

ated

smal

l_G

TPas

eW

D40

Pfam

:Alp

ha_

kin

ase

Pfam

:Ion

_tra

ns

Pfam

:Sel

1SE

L1V

WA

Arf

Gap

Pfam

:Bea

chPB

1FB

OX

UM

EPf

am:N

UC

194

Pfam

:HEA

TPf

am:F

ATPf

am:F

ATC

PI3K

cPf

am:T

PR_2

Pfam

:WH

2FY

VE

MIT

PX Pfam

:Fz

KR

IG_l

ike

IGc2

Rh

oG

EFSE

C14

SPEC

Pfam

:YLP

Pfam

:Rec

ep_L

_do

mai

nFU EP

H_l

bd

SAM

FN3

LY Rh

oG

AP

Pfam

:Kel

ch_2

Pfam

:Kel

ch_1

Pfam

:I-se

tPf

am:L

RR_1

IG ARM

Pfam

:EG

F_2

EGF

EGF_

like

WR1

Hr1

C2

C1

PH CN

HPB

DRB

DPf

am:H

R1R

GS

L27

Gu

Kc

PDZ

LIM

B41

Pfam

:Fo

cal_

ATFC

HSH

2SH

3BT

KPf

am:R

ho

GEF

IQ MYS

cSP

RYZ

nF_

ZZ

Pfam

:PB

DPf

am:U

BA

Pfam

:KA

1U

BA

Pfam

:HSP

20Pf

am:U

BA

_2G

SPf

am:A

ctiv

in_r

ecp

Pfam

:Dea

thD

EPPf

am:A

cety

ltra

nsf

_1ca

lpai

n_I

IIPf

am:A

NF_

rece

pto

rC

YCc

TUD

OR

Pfam

:RIO

1FA

58C

RIO

HA

MP

PAC

Pfam

:CH

ASE

HAT

Pase

_cH

isK

ARE

CPA

SA

AA

GA

FFH

APf

am:C

aMK

II_A

DPT

Pc_D

SPc

Dn

aJSP

KA

DF

TBCK_HsCG4041_DmTBCK_MmC33F10.2_CeTBCK_Ddunc-43_CeCaMK2d_HsCaMK2g_MmCaMK2a_MmCaMK2g_HsCaMK2b_HsCaMK2a_HsRIOK1_HsRio1_DdRIOK3_HsZK632.3_CeRIO1_ScCG11660_DmRIOK3_MmRIO2_ScRIOK1_MmM01B12.5_CeCG3008_Dmauxillin_DmGAK_HsGAK_MmBMPR1B_MmALK2_MmALK7_MmTGFbR1_HsALK1_HsBMPR1A_Mmsma-6_CeALK1_MmSAX_DmBABO_DmBMPR1B_HsALK4_HsALK7_HsALK4_MmALK2_Hsdaf-1_CeTGFBR1_Mmtkv_DmBMPR1A_HsACTR2B_MmACTR2_HsBMPR2_MmACTR2_MmACTR2B_Hsput_DmRYK_Mmdrl_DmRYK_Hslin-18_Cednt_DmPLK1_Mmplk-1_CePLK3_HsPLK3_MmF55G1.8_CePLK5_Mmplk-2_CeCDC5_ScPLK2_Mmpolo_DmALK_Hsscd-2_CeALK_MmAlk_DmADCK2_MmAdckC_DdADCK3_HsCG3608_DmCG7616_DmAdckB1_DdYLR253W_ScY32H12A.7_CeABC1_ScAdckA_DdADCK3_MmADCK1_HsADCK4_MmD2023.6_CeADCK1_MmADCK2_HsAdckB2_DdR04A9.5_CeRoco8_DdY73B6A.1_CeRET_MmRET_HsCad96Ca_Dmchk-2_CeFHAK1_DdFHAK2_DdMEK1_ScFHAK4_DdFHAK3_Ddlok_DmCHK2_MmRAD53_ScT08D2.7_CeDUN1_ScCHK2_HsFHAK5_DdRIM15_ScDhkF_DdDhkA_DdSLN1_ScDhkK_DdDhkC_DdDhkJ_DdDhkI_C2d_DdDhkI_DdDokA_DdDhkE_DdDhkH_DdDhkB_DdDhkD_DdDhkL_DdDhkM_DdDhkG_DdPDK_DmPDHK3_HsZK370.5_CeBCKDK_MmBCKDK_HsPDHK4_HsPDHK1_MmYIL042C_ScPDHK4_MmPDHK2_MmPDHK1_HsPDHK2_HsPDHK3_MmDDB0231454_DdPASK_MmPASK_HsYOL045W_ScCG3105_DmFUN31_ScGCN2_MmY81G3A.3_CeGCN2_ScGCN2_HsIfkB_DdDDB0216407_DdIfkA_DdGcn2_DmIRAK1_MmIRAK3_MmIRAK4_Mmpik-1_CeDDB0231326_DdC01G12.1_CeMYO3B_MmninaC_DmMYO3A_HsMYO3A_MmMYO3B_HsDDB0229854_DdCG4839_DmPKG2_Mmfor_DmPKG1_MmPKG1_HsC09G4.2_Ceegl-4_CePkg21D_DmPKG2_HsCG1973_DmFused_HsSCYL1_MmFused_MmScy1_DdBcDNA:LD22679_DmSCYL3_MmSCYL1_Hstsunami_DdSCYL3_HsW07G4.3_CePIK3R4_HsVPS15_ScPIK3R4_MmCG9746_DmZK930.1_CeVps15_DdSMG1_DdCG4549_DmDNAPK_HsTOR2_ScTor_DdFRAP_MmTOR1_ScTor_DmTRA1_ScFRAP_HsATM_MmATR_Mmmei-41_DmMEC1_ScATR_DdATR_HsB0261.2_Ceatm-1_Ceatl-1_CeTRRAP_HsDNAPK_MmSMG1_MmTEL1_ScATM_HsTRRAP_DdDNAPK_DdCG6535_Dmsmg-1_CeC47D12.1_CeTRRAP_MmCG2905_DmSMG1_HsLRRK2_MmLRRK2_HsMHCK-A_DdMHCK-B_DdMHCK-C_DdMHCK-D_DdLvsG_DdDDB0230126_DdDDB0229872_DdRoco7_DdAlphaK2_HsAlphaK2_MmChaK1_HsChaK2_MmAlphaK3_MmAlphaK3_Hsefk-1_CeVwkA_Ddak1_DdeEF2K_MmeEF2K_HsGbpC_DdRoco11_DdRoco4_DdRoco5_DdPats1_DdRoco6_DdSLOB1_DdDDB0220701_DdCG8726_DmSGK3_HsSGK3_MmSlob_HsSlob_MmCG7156_DmRSKL1_MmRSKL1_HsULK3_HsULK3_MmRSKL2_HsCG8866_DmRSKL2_MmRoco10_DdDDB0229963_DdRoco9_DdDDB0229972_DdDDB0230038_DdNEK10_MmDDB0230124_DdQkgA_C2d_DdQkgA_DdTRKA_MmTRKC_Mmpqn-25_CeDDB0229848_DdSAMK-B_Ddpll_DmRIPK1_MmRIPK1_HsIRAK3_HsDAPK1_HsK12C11.4_CeDAPK1_MmARCK-1_Ddshark_DmHH498_DdLRRK1_HsSgK288_HsIlk_DmDDB0231195_DdHH498_MmSgK288_MmANKRD3_HsC24A1.3_CeDDB0231196_DdCG5483_DmDDB0229339_DdSgK424_HsDDB0231559_DdHH498_HsILK_HsMAP3K8_MmLRRK1_MmSgK307_Hspat-4_CeILK_MmANKRD3_MmSgK307_MmSgK424_MmRNAseL_HsRNAseL_MmPEK_Dmpek-1_Ceire-1_DmIRE2_MmIRE1_Scire-1_CeIreA_DdIRE1_HsIRE1_MmIRE2_HsNrk_DmRor_DmROR2_HsROR2_MmROR1_HsROR1_MmMUSK_MmMUSK_Hscam-1_CeTIE2_HsTIE2_MmF12F3.2_CeAXL_Hsunc-22_CeStrn-Mlck_DmTYRO3_HsC24G7.5_Cebt_Dmver-2_CeFGFR4_HsTRKC_HsKDR_MmFLT3_MmAlphaK1_MmTRKA_HsFMS_MmFGFR2_MmFLT3_HsAlphaK1_HsTTN_HssmMLCK_HsTYRO3_MmFLT4_MmFLT4_Hshtl_Dmver-3_CeFLT1_MmKDR_Hsver-4_CeKIT_MmPDGFRb_HsFLT1_HsCCK4_MmPDGFRa_HsFGFR3_HsFMS_HsPDGFRb_Mmegl-15_CeCCK4_HsPDGFRa_MmPvr_DmKIT_Hsbtl_Dmotk_DmTRKB_HsFGFR4_MmFGFR1_MmFGFR2_HsTRKB_MmFGFR1_HsFGFR3_MmIRR_MmIGF1R_HsINSR_MmIRR_HsIGF1R_MmINSR_HsInR_Dmdaf-2_Celet-23_CeEgfr_DmEGFR_MmHER4/ErbB4_HsErbB2_MmErbB4_MmHER3/ErbB3_HsHER2/ErbB2_HsEGFR_HsErbB3_MmSAPKalpha_DdSAPKalpha_C2d_DdDDB0229849_DdSTE11_ScZAK_MmZAK_HsR13F6.6_CeSplA_Ddvab-1_CeEph_DmEphB1_MmEphB2_MmEphA8_HsEphB4_MmEphB6_MmEphA8_MmEPHA6_MmEphA5_HsEphA10_HsEphB3_MmEphA7_MmEphA3_HsEphB1_HsEphA1_HsEphA6_HsEphB4_HsEphB3_HsEphB6_HsEphA2_HsEphA4_HsEphA7_HsEphB2_HsEphA10_MmEphA2_MmEphA4_MmEphA3_MmEphA1_MmEphA5_MmMER_MmCG18021_DmMER_HsAXL_MmSPEG_MmsmMLCK_MmSPEG_HsTIE1_HsTIE1_MmROS_Hssev_DmROS_MmC16D9.2_CeC24G6.2_CeTOR_DmWSCK_DmPar-1_Dmpar-1_CeMARK1_MmMARK-A_DdMARK1_HsMARK3_MmMARK4_MmW03G1.6_CeMARK-C_DdKIN2_ScKIN1_ScMARK-B_DdMELK_MmMELK_HsMarkmB3_MmMARK2_HsMarkmB2_MmSIK_HsMarkmD1_MmMarkmA11_MmMarkmB1_MmMARK3_HsMARK4_HsMARK2_MmMarkmA9_MmAck_DmKHS2_MmZC1/HGK_HsKHS1_MmZC2_Mmmig-15_CeHPK1_HsZC2/TNIK_HsGCK_HsZC1_Mmmsn_DmKHS1_HsZC4_MmKHS2_HsZC3/MINK_HsHPK1_MmGCK_MmCG7097_DmZC3_MmZC404.9_CeF19C6.1_CeGPRK6_HsGPRK7_HsRHOK_MmGPRK4_MmGPRK4_HsGPRK5_HsGPRK5_MmRHOK_HsGPRK6_MmGprk2_DmPAK2_HsPAK3_Mmpak-1_CePAK1_MmPAK4_HsPAK1_HsPAK5_MmPAK2_MmPak_DmSTE20_ScPakB_DdPakA_DdPAK4_Mmmbt_DmPAK3_HsPAK5_HsPAK6_MmC45B11.1_CePAK6_HsCLA4_ScPakC_DdSKM1_ScPakD_DdPak3_DmPKCt_Hsksr-2_CePKCdelta_DmDDB0231197_Ddksr-1_CeKSR2_HsKSR2_MmKSR1_MmPKCt_Mmtpa-1_Ceksr_DmKSR1_Hsphl_DmARAF_HsRAF1_Mmlin-45_CeARAF_MmRAF1_HsBRAF_MmBRAF_HsPKCeps_MmPkc98E_DminaC_DmPKCd_Mmpkc-2_CePKCa_HsPKCh_HsPKCeta_MmPKCa_MmPkc53E_DmPKCg_Mmkin-13_CePKCd_HsPKCe_HsPKCg_HsPKCb_HsPKCb_MmPKC1_ScMEKKalpha_DdMAP3K3_MmMAP3K2_MmMAP3K2_HsMAP2K5_MmMAP3K3_HsMAP2K5_HsaPKC_DmPKCz_MmPKCz_HsPKCi_Mmpkc-3_CePKCi_HsDDB0229867_DdROCK1_MmROCK1_HsROCK2_Mmrok_DmPKD3_HsPKD2_HsCG7125_DmT25E12.4_CeDMPK2_Mmlet-502_CeW09C5.5_CeDMPK2_HsROCK2_HsPKD2_MmPKD3_MmPKD1_MmPKD1_HsK08B12.5_CeMRCKa_MmMRCKa_HsMRCKb_HsMRCKb_Mmgek_DmCRIK_HsCRIK_MmW02B3.2_CeBARK2_MmBARK2_HsGprk1_DmBARK1_MmBARK1_HsPdk1A_DdAkt1_Dmpdk-1_CeAKT1_MmAKT1_HsAKT3_HsAkt1_DdDDB0229957_Ddakt-2_Ceakt-1_CeAKT3_MmAKT2_HsAKT2_MmDDB0229973_DdBCR_MmBCR_HsPKN3_MmPKN1_HsPKN3_HsPKN2_MmPKN1_MmF46F6.2_CePKN2_HsDDB0220670_DdSCH9_ScBMX_HsBMX_MmITK_HsTEC_HsBTK_HsTEC_MmITK_MmBTK_MmF26E4.5_CeC55C3.4_CeT25B9.4_CeSYK_MmC18H7.4_CeF23C8.7_CeT21G5.1_CeR05H5.4_CeY69E1A.3_CeaSWK345_CeShk1_DdY4C6A.1_CeF59A3.8_CeZAP70_HsShk2_Ddkin-26_CeT25B9.5_CeW01B6.5_CeW02A2.4_CeR11E3.1_CeC34F11.5_CeCG17309_Dmspe-8_CeY52D5A.2_CeaSWK377_CeZK593.9_CeShk4_DdF57B9.8_CeF46F5.2_Cekin-28_CeK09B11.5_CeT06C10.3_Cekin-21_CeW03F8.2_CeZK622.1_CeSYK_HsM176.9_CeShk5_DdC25A8.5_CeC35E7.10_Cekin-5_CeZC581.7_CeShk3_DdZAP70_Mmkin-24_Cekin-14_CeY116A8C.24_Cefrk-1_CeF22B3.8_CeF01D4.3_CeFGR_HsCTK_MmYES_Mmabl-1_CeCSK_MmFRK_HsBLK_MmABL_HsBRK_MmARG_MmAbl_DmBtk29A_DmTXK_MmLYN_MmARG_HsCSK_HsFGR_MmTXK_HsLCK_HsY48G1C.2_CeBLK_HsLYN_HsFYN_HsSRM_MmSRC_HsHCK_MmABL_MmHCK_HsSrc42A_DmSrc64B_DmBRK_HsSRC_MmCTK_HsY47G6A.5_CeFYN_Mmkin-22_CeLCK_MmFRK_Mmsrc-1_CeSRM_HsYES_HsFER_MmFER_HsFES_MmFES_HsFps85D_DmFAK_MmFAK_HsFak56D_DmPYK2_HsTYK2_HsJAK2_Mmhop_DmJAK1_HsTYK2_MmJAK3_MmJAK1_MmJAK3_HsJAK2_Hskin-31_CePYK2_MmCaki_Dmlin-2_CeCASK_HsCASK_MmACK_MmMLK1_MmK11D12.10_CeMLK1_Hskin-25_Ceslpr_DmMLK4_MmTNK1_MmMkcF_DdMLK2_MmMLK3_HsTNK1_HsMLK4_HsACK_HsMLK3_MmMLK2_HsPR2_Dmark-1_CeObscn_MmObscn_HsTrad_HsTrad_MmTrio_MmTrio_HsDDB0229940_DdDDB0218878e_DdMAST4_HsCG6498_DmMAST1_MmMAST1_HsMAST4_Mmkin-4_CeMAST3_HsMAST2_MmMAST2_HsMAST3_MmLIMK2_HsLIMK2_MmLIMK1_MmLIMK1_HsLIMK1_DmLATS1_HsLATS2_HsLATS2_MmLATS1_MmMAP3K1_HsMAP3K1_MmDDB0230051_DdTIF1b_MmTIF1b_HsTIF1g_MmTIF1a_HsTIF1a_MmTIF1g_HsDDB0220714_DdTaf250_Dmtaf-1_CeDDB0220693_DdBRD3_HsY119C1B.8_CeTAF1L_HsBRDT_HsBRD4_HsBRDT_MmTAF1_Mmfs(1)h_DmTAF1_DdBRD2_HsTAF1_HsBRD2_MmBRD4_MmBRD3_MmDDB0220694_DdTAF1_ScFASTK_HsFASTK_MmRON_MmMET_MmMET_HsRON_HsW05B2.1_CeDDB0216331_DdDDB0229344_DdDCAMKL2_MmCG10177_Dmzyg-8_CeDCAMKL2_HsDCAMKL1_MmCG17528_DmDCAMKL1_HsSNF1_ScBUB1_ScBub1_DmCG14030_DmBUBR1_HsBUB1_HsBub1_DdBUB1_MmBUBR1_MmH11_HsH11_MmSgK396_MmSgK396_HsKIS_HsKIS_MmRIPK2_MmRIPK2_HsY39G8B.5_CeFNIPK-C_DdFNIPK-D_DdFNIPK-A_DdFNIPK-E_DdFNIPK-D_C2d_DdDDB0231199_DdDDB0216375_DdPKR_HsPKR_MmC25F6.4_CeCG11573_DmDDR1_MmDDR2_HsF11D5.3_CeDDR1_HsDDR2_Mmtwf_Dmunc-60_CeA6_MmA6r_MmF38E9.5_CeA6r_HsA6_HsTWF1_Scgcy-27_Ceodr-1_Cegcy-25_Cegcy-23_Cegcy-21_Cegcy-17_CeCG4224_DmCYGX_Mmgcy-19_Cegcy-15_CeT01A4.1_Cegcy-11_CeCG3216_DmC04H5.4_CeKSGC_MmGyc32E_Dmdaf-11_CeHSER_MmCG9783_Dmgcy-7_Cegcy-14_CeHSER_HsCYGF_HsCYGD_Mmgcy-18_Cegcy-6_Cegcy-13_CeCG10738_Dmgcy-12_Cegcy-3_Cegcy-8_CeANPa_MmANPb_Mmgcy-22_CeANPb_HsCYGD_HsANPa_Hsgcy-5_CeCYGF_Mmgcy-20_Cegcy-9_Cegcy-1_CeGyc76C_Dmgcy-2_Cegcy-4_CeC16A11.3_CeH12I13.1_CeCG10951_DmNEK8_MmNEK9_MmNEK9_HsDDB0219986_DdTBK1_MmPRPK_HsTBK1_HsIKKe_HsaSWK440_CeRio2_DdCG11859_DmRIOK2_MmCG10673_DmRIOK2_Hs

HsMmDmCeDdSc


FCH

SH

2S

H3

BTK

BMX_HsBMX_MmITK_HsTEC_HsBTK_HsTEC_MmITK_MmBTK_MmF26E4.5_CeC55C3.4_CeT25B9.4_CeSYK_MmC18H7.4_CeF23C8.7_CeT21G5.1_CeR05H5.4_CeY69E1A.3_CeaSWK345_CeShk1_DdY4C6A.1_CeF59A3.8_CeZAP70_HsShk2_Ddkin-26_CeT25B9.5_CeW01B6.5_CeW02A2.4_CeR11E3.1_CeC34F11.5_CeCG17309_Dmspe-8_CeY52D5A.2_CeaSWK377_CeZK593.9_CeShk4_DdF57B9.8_CeF46F5.2_Cekin-28_CeK09B11.5_CeT06C10.3_Cekin-21_CeW03F8.2_CeZK622.1_CeSYK_HsM176.9_CeShk5_DdC25A8.5_CeC35E7.10_Cekin-5_CeZC581.7_CeShk3_DdZAP70_Mmkin-24_Cekin-14_CeY116A8C.24_Cefrk-1_CeF22B3.8_CeF01D4.3_CeFGR_HsCTK_MmYES_Mmabl-1_CeCSK_MmFRK_HsBLK_MmABL_HsBRK_MmARG_MmAbl_DmBtk29A_DmTXK_MmLYN_MmARG_HsCSK_HsFGR_MmTXK_HsLCK_HsY48G1C.2_CeBLK_HsLYN_HsFYN_HsSRM_MmSRC_HsHCK_MmABL_MmHCK_HsSrc42A_DmSrc64B_DmBRK_HsSRC_MmCTK_HsY47G6A.5_CeFYN_Mmkin-22_CeLCK_MmFRK_Mmsrc-1_CeSRM_HsYES_HsFER_MmFER_HsFES_MmFES_HsFps85D_DmFAK_MmFAK_HsFak56D_DmPYK2_HsTYK2_HsJAK2_Mmhop_DmJAK1_HsTYK2_MmJAK3_MmJAK1_MmJAK3_HsJAK2_Hs

Pfa

m:F

zK

RIG

_lik

eIG

c2R

hoG

EF

SE

C14

SP

EC

Pfa

m:Y

LPP

fam

:Rec

ep_L

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ain

FU EP

H_l

bdS

AM

FN3

LY

Nrk_DmRor_DmROR2_HsROR2_MmROR1_HsROR1_MmMUSK_MmMUSK_Hscam-1_CeTIE2_HsTIE2_MmF12F3.2_CeAXL_Hsunc-22_CeStrn-Mlck_DmTYRO3_HsC24G7.5_Cebt_Dmver-2_CeFGFR4_HsTRKC_HsKDR_MmFLT3_MmAlphaK1_MmTRKA_HsFMS_MmFGFR2_MmFLT3_HsAlphaK1_HsTTN_HssmMLCK_HsTYRO3_MmFLT4_MmFLT4_Hshtl_Dmver-3_CeFLT1_MmKDR_Hsver-4_CeKIT_MmPDGFRb_HsFLT1_HsCCK4_MmPDGFRa_HsFGFR3_HsFMS_HsPDGFRb_Mmegl-15_CeCCK4_HsPDGFRa_MmPvr_DmKIT_Hsbtl_Dmotk_DmTRKB_HsFGFR4_MmFGFR1_MmFGFR2_HsTRKB_MmFGFR1_HsFGFR3_MmIRR_MmIGF1R_HsINSR_MmIRR_HsIGF1R_MmINSR_HsInR_Dmdaf-2_Celet-23_CeEgfr_DmEGFR_MmHER4/ErbB4_HsErbB2_MmErbB4_MmHER3/ErbB3_HsHER2/ErbB2_HsEGFR_HsErbB3_MmSAPKalpha_DdSAPKalpha_C2d_DdDDB0229849_DdSTE11_ScZAK_MmZAK_HsR13F6.6_CeSplA_Ddvab-1_CeEph_DmEphB1_MmEphB2_MmEphA8_HsEphB4_MmEphB6_MmEphA8_MmEPHA6_MmEphA5_HsEphA10_HsEphB3_MmEphA7_MmEphA3_HsEphB1_HsEphA1_HsEphA6_HsEphB4_HsEphB3_HsEphB6_HsEphA2_HsEphA4_HsEphA7_HsEphB2_HsEphA10_MmEphA2_MmEphA4_MmEphA3_MmEphA1_MmEphA5_MmMER_MmCG18021_DmMER_HsAXL_MmSPEG_MmsmMLCK_MmSPEG_HsTIE1_HsTIE1_MmROS_Hssev_DmROS_MmC16D9.2_CeC24G6.2_CeTOR_DmWSCK_Dm

Hr1

C2

C1

PH CN

HPB

DRB

DPf

am:H

R1R

GS

PB1

FBO

X

KHS2_MmZC1/HGK_HsKHS1_MmZC2_Mmmig-15_CeHPK1_HsZC2/TNIK_HsGCK_HsZC1_Mmmsn_DmKHS1_HsZC4_MmKHS2_HsZC3/MINK_HsHPK1_MmGCK_MmCG7097_DmZC3_MmZC404.9_CeF19C6.1_CeGPRK6_HsGPRK7_HsRHOK_MmGPRK4_MmGPRK4_HsGPRK5_HsGPRK5_MmRHOK_HsGPRK6_MmGprk2_DmPAK2_HsPAK3_Mmpak-1_CePAK1_MmPAK4_HsPAK1_HsPAK5_MmPAK2_MmPak_DmSTE20_ScPakB_DdPakA_DdPAK4_Mmmbt_DmPAK3_HsPAK5_HsPAK6_MmC45B11.1_CePAK6_HsCLA4_ScPakC_DdSKM1_ScPakD_DdPak3_DmPKCt_Hsksr-2_CePKCdelta_DmDDB0231197_Ddksr-1_CeKSR2_HsKSR2_MmKSR1_MmPKCt_Mmtpa-1_Ceksr_DmKSR1_Hsphl_DmARAF_HsRAF1_Mmlin-45_CeARAF_MmRAF1_HsBRAF_MmBRAF_HsPKCeps_MmPkc98E_DminaC_DmPKCd_Mmpkc-2_CePKCa_HsPKCh_HsPKCeta_MmPKCa_MmPkc53E_DmPKCg_Mmkin-13_CePKCd_HsPKCe_HsPKCg_HsPKCb_HsPKCb_MmPKC1_ScMEKKalpha_DdMAP3K3_MmMAP3K2_MmMAP3K2_HsMAP2K5_MmMAP3K3_HsMAP2K5_HsaPKC_DmPKCz_MmPKCz_HsPKCi_Mmpkc-3_CePKCi_HsDDB0229867_DdROCK1_MmROCK1_HsROCK2_Mmrok_DmPKD3_HsPKD2_HsCG7125_DmT25E12.4_CeDMPK2_Mmlet-502_CeW09C5.5_CeDMPK2_HsROCK2_HsPKD2_MmPKD3_MmPKD1_MmPKD1_HsK08B12.5_CeMRCKa_MmMRCKa_HsMRCKb_HsMRCKb_Mmgek_DmCRIK_HsCRIK_MmW02B3.2_CeBARK2_MmBARK2_HsGprk1_DmBARK1_MmBARK1_HsPdk1A_DdAkt1_Dmpdk-1_CeAKT1_MmAKT1_HsAKT3_HsAkt1_DdDDB0229957_Ddakt-2_Ceakt-1_CeAKT3_MmAKT2_HsAKT2_MmDDB0229973_DdBCR_MmBCR_HsPKN3_MmPKN1_HsPKN3_HsPKN2_MmPKN1_MmF46F6.2_CePKN2_HsDDB0220670_DdSCH9_Sc

RIM15_ScDhkF_DdDhkA_DdSLN1_ScDhkK_DdDhkC_DdDhkJ_DdDhkI_C2d_DdDhkI_DdDokA_DdDhkE_DdDhkH_DdDhkB_DdDhkD_DdDhkL_DdDhkM_DdDhkG_DdPDK_DmPDHK3_HsZK370.5_CeBCKDK_MmBCKDK_HsPDHK4_HsPDHK1_MmYIL042C_ScPDHK4_MmPDHK2_MmPDHK1_HsPDHK2_HsPDHK3_MmDDB0231454_DdPASK_MmPASK_HsYOL045W_ScCG3105_DmFUN31_Sc

HA

MP

PA

CP

fam

:CH

AS

EH

ATP

ase_

cH

isK

AR

EC

PA

SA

AA

GA

FFH

A

CG1973_DmFused_HsSCYL1_MmFused_MmScy1_DdBcDNA:LD22679_DmSCYL3_MmSCYL1_Hstsunami_DdSCYL3_HsW07G4.3_CePIK3R4_HsVPS15_ScPIK3R4_MmCG9746_DmZK930.1_CeVps15_DdSMG1_DdCG4549_DmDNAPK_HsTOR2_ScTor_DdFRAP_MmTOR1_ScTor_DmTRA1_ScFRAP_HsATM_MmATR_Mmmei-41_DmMEC1_ScATR_DdATR_HsB0261.2_Ceatm-1_Ceatl-1_CeTRRAP_HsDNAPK_MmSMG1_MmTEL1_ScATM_HsTRRAP_DdDNAPK_DdCG6535_Dmsmg-1_CeC47D12.1_CeTRRAP_MmCG2905_DmSMG1_HsLRRK2_MmLRRK2_HsMHCK-A_DdMHCK-B_DdMHCK-C_DdMHCK-D_DdLvsG_DdDDB0230126_DdDDB0229872_DdRoco7_DdAlphaK2_HsAlphaK2_MmChaK1_HsChaK2_MmAlphaK3_MmAlphaK3_Hsefk-1_CeVwkA_Ddak1_DdeEF2K_MmeEF2K_Hs

smal

l_G

TPas

eW

D40

Pfam

:Alp

ha_

kin

ase

Pfam

:Ion

_tra

ns

Pfam

:Sel

1

UM

EPf

am:N

UC

194

Pfam

:HEA

TPf

am:F

ATPf

am:F

ATC

PI3K

cPf

am:T

PR_2

PQ

QP

UG

Pfa

m:S

AM

_1P

fam

:SA

M_2

AN

KD

EA

TH

DDB0229848_DdSAMK-B_Ddpll_DmRIPK1_MmRIPK1_HsIRAK3_HsDAPK1_HsK12C11.4_CeDAPK1_MmARCK-1_Ddshark_DmHH498_DdLRRK1_HsSgK288_HsIlk_DmDDB0231195_DdHH498_MmSgK288_MmANKRD3_HsC24A1.3_CeDDB0231196_DdCG5483_DmDDB0229339_DdSgK424_HsDDB0231559_DdHH498_HsILK_HsMAP3K8_MmLRRK1_MmSgK307_Hspat-4_CeILK_MmANKRD3_MmSgK307_MmSgK424_MmRNAseL_HsRNAseL_MmPEK_Dmpek-1_Ceire-1_DmIRE2_MmIRE1_Scire-1_CeIreA_DdIRE1_HsIRE1_MmIRE2_Hs

MAP3K1_HsMAP3K1_MmDDB0230051_DdTIF1b_MmTIF1b_HsTIF1g_MmTIF1a_HsTIF1a_MmTIF1g_HsDDB0220714_DdTaf250_Dmtaf-1_CeDDB0220693_DdBRD3_HsY119C1B.8_CeTAF1L_HsBRDT_HsBRD4_HsBRDT_MmTAF1_Mmfs(1)h_DmTAF1_DdBRD2_HsTAF1_HsBRD2_MmBRD4_MmBRD3_MmDDB0220694_Dd

PH

DB

BC

BB

OX

RIN

GB

RO

MO

Kel

chA

T_ho

okZn

F_C

2HC

PS

IIP

TS

ema

RON_MmMET_MmMET_HsRON_Hs

Pfa

m:R

AP

Pfa

m:F

AS

T_2

Pfa

m:F

AS

T_1

FASTK_HsFASTK_Mm

a

a

b

b

c

c d

d

e

f

f

e

g

g

hhi

i

domain

protein

fig. S4. Domain profiling across six eukaryotic kinomes

Amalgamated kinomes of six eukaryotic species of Saccharomyces cerevisiae (Sc),

Dictyostelium discoideum (Dd), Caenorhabditis elegans (Ce), Drosophila melanogaster

(Dm), Mus musculus (Mn) and Homo sapiens (Hs) were clustered through their non-

catalytic domains (similar to S3, lower panel). kinase sequences and nomenclatures were

adapted from kinase.com; pixel color indicates species. To the right: Magnified version

of selected clusters (boxes a-i). The great majority of proteins in clusters a and b

(indicated by the red box) are tyrosine kinases (TK).

0.563

0.653

0.554

0.7030.563

0.643

0.5

0.333

0.5

0.333

0.352

0.3380.447

0.325

0.434

0.2

0.289

0.378

0.20.312

0.25

00.8 0.6 0.6 0.210

0.10.20.30.40.50.60.70.80.9

1

Similarity coefficient index (Jaccard's)

Com

pact

fact

or

(# o

f clu

bs /

tota

l # o

f pro

tein

s)

cut-off point


Domain club #53:

BBOXBBOX BROMOBROMO

BBOXBBOX BROMOBROMO

Domain club #54:

PHDPHD BROMOBROMO PWWPPWWP

PWWPPWWP

BROMOBROMO PWWPPWWP

Domain club #55:

PHDPHD BROMOBROMOSANDSAND

PHDPHD BROMOBROMOAT_hookAT_hook DDTDDT

PHDPHD BROMOBROMO

PHDPHD BROMOBROMODDTDDTMBDMBD

PHDPHD BROMOBROMODDTDDT

BBCBBC PHDPHD

RINGRING BBCBBC

Zf_C2H2Zf_C2H2

RINGRING

PHDPHDBBCBBCRINGRING

BROMOBROMO

PHDPHD

BBOXBBOX BROMOBROMO

MBDMBD

Domain club #56:BROMOBROMO

BROMOBROMOWD40WD40

Domain club #420:

SH2SH2SH2SH2PTBPTB

PTBPTBPTPc_DSPcPTPc_DSPc

PTPc_DSPcPTPc_DSPc

C1C1

Domain club #421:

SH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

SOCSSOCS

Domain club #422:

SH2SH2SH2SH2

SH2SH2SH2SH2

RhoGAPRhoGAP

Domain club #423:

SH2SH2SH2SH2 TyrKcTyrKc

SH3SH3

FCHFCH

SH3SH3

SH3SH3



PTBPTBSH2SH2SH2SH2

SH2SH2SH2SH2 SH3SH3 SH3SH3

SH2SH2SH2SH2SH3SH3 SH3SH3 SH3SH3

SH2SH2SH2SH2

PTPcPTPc

SH2SH2SH2SH2SH2SH2SH2SH2 PTPcPTPc

PTBPTBSH2SH2SH2SH2

SH2SH2SH2SH2

BBOXBBOX BROMOBROMO

BBOXBBOX BROMOBROMO


PWWPPWWP

BROMOBROMO PWWPPWWP



PHDPHD BROMOBROMO



BBCBBC PHDPHD

RINGRING BBCBBC

Zf_C2H2Zf_C2H2

RINGRING


BROMOBROMO

PHDPHD

BBOXBBOX BROMOBROMO

MBDMBD

BROMOBROMO

BROMOBROMOWD40WD40

SH2SH2SH2SH2PTBPTB


PTPc_DSPcPTPc_DSPc

C1C1

SH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

SOCSSOCS

SH2SH2SH2SH2

SH2SH2SH2SH2

RhoGAPRhoGAP


SH3SH3

FCHFCH

SH3SH3

SH3SH3



PTBPTBSH2SH2SH2SH2



SH2SH2SH2SH2

PTPcPTPc


PTBPTBSH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

RhoGAPRhoGAP


SH3SH3

FCHFCH

SH3SH3

SH3SH3





SH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

SH2SH2SH2SH2

SOCSSOCS

SH2SH2SH2SH2PTBPTB


PTPc_DSPcPTPc_DSPc

C1C1

SH2SH2SH2SH2

PTBPTBSH2SH2SH2SH2

PTPcPTPc


PTBPTBSH2SH2SH2SH2

BBOXBBOX BROMOBROMO

BBOXBBOX BROMOBROMO


PWWPPWWP

BROMOBROMO PWWPPWWP



PHDPHD BROMOBROMO



BBCBBC PHDPHD

RINGRING BBCBBC

Zf_C2H2Zf_C2H2

RINGRING


BROMOBROMO

PHDPHD

BBOXBBOX BROMOBROMO

MBDMBD

BROMOBROMO

BROMOBROMOWD40WD40

0.4

fig. S5. Domain club cut-off selection

For every possible value of the domain club cut-off from the 7 genome co-clustering, we

computed the number of resulting domain clubs, and plotted this number as a function of

the cut-off (plot panel). The figure shows this function, as well as the chosen cut-off

(similarity coefficient index at 0.502 - indicated by an arrow). The chosen cut-off

corresponds to a natural jump in the plotted curve (plot panel), resulting in 1,245 clubs

(#1-1,245, Figure 3A). At this cut-off (illustrated by a black dashed line), example

domain clubs (#53-56, and #420-423, as in Figure 3A) are shown in the foreground (solid

boxes, center panels). The branching patterns of the tree in these two areas are also

mapped (solid lines, values for the similarity coefficient marked at branches). The

groupings at the various cut-offs (corresponding to the dashed lines) are shown in the

background (dashed boxes in fading colors). Note that at a lower cut-off (right panels)

when proteins that only contain an SH2 domain form a club, this remains on the same

branch of the tree and is surrounded by the same neighbourhood of domain compositions.

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Dicer1

similarity index in seven-proteome

sim

ilarit

y in

dex

in n

ine-

prot

eom

e

r : 0.9975p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

ZMYND11


sim

ilarit

y in

dex

in n

ine-

prot

eom

e

r : 0.9444p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Tdrd1


sim

ilarit

y in

dex

in n

ine-

prot

eom

r : 0.9219p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

7-proteome

Fes

Dic

er1

r :-0.0204p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Fes


r : 0.9566p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

JMJD2C


r : 0.9363p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

EGFR


r : 0.9593p < 0.001

0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

9-proteome

Fes

r :-0.0174p < 0.001

A

B


fig. S6. Test of the robustness of the domain profiling method for applications in

large data sets

A. We made a direct comparison of the results from clustering of seven proteomes

(Figure 3) with those from another amalgamated set containing two additional proteomes,

those of Xenopus tropicalis and Mus musculus (nine-proteome). Specifically, we selected

six protein examples (Dicer1, Fes, ZMTND11, JMJD2C, Tdrd1 and EGFR) involved in

various biological processes, and for each protein, computed its distance to every other

protein in the seven-proteome protein set (total number of proteins exceeding 40,000) and

in the nine-proteome protein set. The distance pairs (in fact, the equivalent similarity

pairs) are represented in each plot as circles whose x-coordinates are measurements in

seven-proteome protein set and y-coordinates are the measurements in the nine-proteome

protein set. We note a consistent distribution pattern along the y=x line in every plot,

which is indicative of positive correlations between x- and y-axis values. Since the

majority of the measurement pairs are densely located in the upper-right corner in each

graph (0.95-1.0 along both x- and y-axes) and details of such correlations are not visually

apparent, we in addition calculated the Pearson’s Correlation Coefficient (r) to relate the

x- and y-axes values. For all six examples, strong positive correlations are observed (r-

values exceeding 0.9, in red), indicating the consistency between the tree-grams resulting

from datasets of different sizes (seven and nine proteomes). Also given in the plots are p-

values against the null hypotheses that the measurements in different-sized datasets be

completely unrelated.

B. In a negative control analysis, two protein examples (Dicer1 and Fes, residing in club

#188 and club #423 respectively) sampled from distinct branches of the tree (Figure 3A,

S6, and supplementary section S-III) are compared. In the plots, the y-coordinate is the

similarity between Dicer1 and every other protein in the protein set, and the x-coordiate

is the Fes counterpart. In both the seven-proteome and the nine-proteome plots, this

yielded distinct distribution patterns from those shown in A, and in both instances, a lack

of correlation indicated by small r-values (-0.0204 and -0.0174 respectively, in blue) was

observed. Such results indicate that distant protein pairs in the proteomes (i.e. Dicer and

Fes) would consistently remain separated from each other following domain profiling,

and this is not affected by the size of dataset.

Domain Clubs (1 to 1245)

DICER1

..

A


B

HELICc Domain

200 400 600 800 1000 1200

Sim

ilarit

yIn

dex

Yea

stS

lime

Mol

dW

orm

Frui

t Fly

Zebr

afis

hC

hick

enH

uman

Num

ber o

f Pro

tein

s

1.00

0.98

0.96

0

103

51

0

73

36

0

84

42

0

70

35

0

81

40

0

26

13

0

72

36

DSRM Domain

200 400 600 800 1000 1200

Sim

ilarit

yIn

dex

Yea

stS

lime

Mol

dW

orm

Frui

t Fly

Zebr

afis

hC

hick

enH

uman

Num

ber o

f Pro

tein

s

1.00

0.98

0.96

0

9

4

0

6

3

0

8

4

0

8

4

0

6

3

0

10

2

..Club #180

DGCR8* WWDSRM

DSRM

DUS2L Pfam:DusDSRM

EIF2AK2 DSRMDSRM

STYKc

ILF3 DZFDSRM

DSRM

PRKRA DSRMDSRM

DSRM

STAU1* DSRMDSRM

DSRM

STAU2 DSRMDSRM

DSRMDSRM

STRBP DZFDSRM

DSRM

TARBP2 DSRMDSRM

DSRM

Club #181

ADAD1 DSRMADEAMc

ADAR ZalphaZalpha

DSRMDSRM

DSRMADEAMc

ADARB1 DSRMDSRM

ADEAMc

ADARB2 DSRMDSRM

ADEAMc

Club #188

DHX9* DSRMDSRM

DEXDcHELICc

HA2Pfam:DUF1605

DICER1* DEXDcHELICc

Pfam:dsRNA_bindPfam:PAZ

RIBOcRIBOc

DSRM

Club #190

NKRF DSRMDSRM

G_patchR3H

Club #265

SLC4A1AP FHADSRM

CCCC

Club #458

RNASEN/DROSHA* RIBOcRIBOc

DSRM

DEXDcHELICc

Pfam:dsRNA_bind

Pfam:PAZRIBOc RIBOc

DSRM

DSRM domainDSRM

fig. S7. A website for domain club analysis – example of Dicer1 and its DSRM

domain

Panel A. A keyword query for Dicer (protein) directs the reader to a page that displays

domain club profiles across seven eukaryotic proteomes (similar to the panels in Figure

3B), for each of Dicer’s domains (SMART definition), namely DEXDc, HELICc, DSRM

and RIBOc. The profiles for HELICc and DSRM are shown. The top panel shows all the

clubs that contain HELICc domains, and the height of the rod indicates the number of

proteins in these clubs with HELICc domains. Similarly, the lower panel indicates all of

the clubs that contain the DSRM domain, and the height of the rod indicates the total

number of proteins in these clubs with DSRM domains. The domain club containing

Dicer1 is labeled in red. It is interesting to note that DSRM (and RIBOc, not shown)

domains are absent from the “red” club (#188) of yeast and slime mold.

A query for the DSRM domain displays the relevant domain club profiles (shown at the

bottom of panel A) and the domain architectures of all human proteins containing the

DSRM domain (panel B). In yeast and slime mold, there is only one DSRM domain club,

which is occupied by the more ancient RNASEN/Ribonuclease III proteins (dashed boxes

in A, bottom panel and B) and an atypical slime mold Dicer-like protein that, like

RNASEN, has only DSRM and RIBOc domains. The metazoan Drosha protein has the

same domain combination as RNASEN, and is therefore found in club #458. The results

indicate that the DSRM domain has expanded to new domain clubs (#180, 181, 188, 190

and 265; asterisks mark proteins with known functions in small non-coding RNA

biology.) in metazoan species (arrow).

Protein A

Protein B

A

Protein C

Domain Y

Domain Y

Domain X

Domain X

Dom

ain

XDo

main

Y

... ........ ................. ..

..........

.........

... ... ....

...... ..........

.

..... ............

.Spreading (refined)

1 2

1 3

1 4

Relatedness

+

=+

=

+

=

1 1 13 3 3

1

2

4

3

Spreading (simple)

Domain

Pro

tein

Domain

Pro

tein

Domain

B

Density (height) survey*

* Density plot on matrix (visualization) and vector scanning of density elements between density maps (computing correlations)

Spreading Spreading with decay (smoothing)

verticalview

horizontalview

Molecular environment(niche) for domain X

Domain X -containing protein

Bachground: whole proteome domain clustergramPixels on the grid, right panel: Molecular environment for domain X family


fig. S8. General procedure for spreading-on-graph (SOG) clustering in the analysis

of the molecular environments of domain families

A. The molecular environment of a domain family, say X, is the set of all proteins that

reportedly interact with domain X (direct interaction) or with coexisting domains/motifs

on the same polypeptide chain as domain X (associated interactions). B. As a template to

plot such interactions, we generated a protein-domain clustergram of the human proteome

(schematized in B, left panel), and scored those proteins on the matrix that interact with

proteins containing domain X, yielding a scatter pattern that represents the molecular

environment of domain X (B, right panel). The molecular environments of multiple

domains are then compared by spreading on graph (SOG) clustering of their respective

scatter patterns. In SOG clustering, the impact of each point in the scatter pattern is

spread to neighbouring areas in the clustergram to reflect the relatedness of the pixels (B

right), yielding a smoothed distribution of density (represented by height). Most simply,

spreading extends the impact of a point uniformly to coordinates within a fixed distance.

This approach may be refined by spreading the impact of a point to all other coordinates

non-uniformly following a Gaussian function of distance.

SH3

RIN

GSH

2P

H C2

PD

ZW

D40 C

1A

NK EF

BR

OM

O14

_3_3

LRR

UB

AC

HW

WB

41 LIM

DE

ATH

TPR

AR

MM

H1

MH

2C

HR

OM

OB

TBS

AM

PTB

RH

OG

EF

BR

CT

HE

CT

FHA

PB

1TR

AF

PA

SR

GS

CA

RD IQ

TUD

OR PX

FYV

ED

SH

UIM

DED

F_B

OX

TIR

WH

1B

H4

VH

SE

HT_

SN

AR

ES

OC

SH

EA

TB

AR

CU

EB

IRP

OLO

_BO

XG

EL

AD

FE

NTH

GA

TFH

2V

HL

STA

RT

GR

AM FF

TUB

PU

MIL

IOG

YF

BE

AC

HG

RIP

0

200

400

600

800

1000

1200

0

50

100

150

200

250

SH3

RIN

GSH

2P

H C2

PD

ZW

D40 C1

AN

K EF

BR

OM

O14

_3_3

LRR

UB

AC

HW

WB

41 LIM

DE

ATH

TPR

AR

MM

H1

MH

2C

HR

OM

OB

TBS

AM

PTB

RH

OG

EF

BR

CT

HE

CT

FHA

PB

1TR

AF

PA

SR

GS

CA

RD IQ

TUD

OR PX

FYV

ED

SH

UIM

DED

F_B

OX

TIR

WH

1B

H4

VH

SE

HT_

SN

AR

ES

OC

SH

EA

TB

AR

CU

EB

IRP

OLO

_BO

XG

EL

AD

FE

NTH

GA

TFH

2V

HL

STA

RT

GR

AM FF

TUB

PU

MIL

IOG

YF

BE

AC

HG

RIP#

of d

omai

n-co

ntai

ning

pro

tein

s#

of in

tera

ctin

g pr

otei

ns


fig S9. The interactomes of the 70 domain families employed in the analysis of domain-based functional compartmentsThe panels plot the number of interacting proteins reported in HPRD for each studied domain family (top, the solid bars correspond to the heat map examples in Figure 4B) and the total number of proteins (in the human proteome) containing each studied domain family (bottom).

other domainPostSET

SET

BRC T

G_patch

KH

BRIGH T

Pfam:zfC2HC

Pfam:MR G

RING

Pfam:MutS_I

PHD

Pfam:HA2

Pfam:zfCW

HM G

SAM

UBA(elongation...)

Pfam:DNA_methylase

Pfam:Myosin_N

Pfam:MutS_II

MUTSac

ChSh

Pfam:NAD_binding_2

JmjC

Pfam:RBB1NT

Pfam:TC H

Pfam:DUF1087

JmjN

Pfam:MOZ_SAS

ZnF_C2H2

Pfam:PreSET

Pfam:CHDCT2

Pfam:Pkinase

MBD

SANT

Pfam:CHDNT

ANK

Pfam:ECH

ZnF_C3H1

AT_hook

Pfam:MBD

DEXD c

IQ

MYSc

Pfam:Shikimate_D H

Pfam:zfMYND

BROMO

Pfam:SWIR M

AWS

PTX

BRK

Pfam:DUF1295

Pfam:ERG4_ERG24

HELICc

PreSET

Pfam:Myosin_tail_1

VWA

Pfam:DUF1086

SNc

MUTSd

Pfam:F420_oxidored

Tudor ENSG198639 1

Tudor TP53BP1 1

Tudor FXR2 1

Tudor SETDB1 2

Tudor RNF17 3

Tudor TDRD7 2

Tudor STK31 1Tudor RNF17 4

Tudor TDRD6 5Tudor SND1 1

Tudor TDR

D1 1

Tudor TDR

D3

1

Tudor SMN

1 1Tudor SM

N2 1

Tudor SMN

1

Tudor SMN

DC

1 1

Tudo

r TD

RD

6 4

Tudo

r E

NS

G19

8639

4

Tudo

r TD

RKH

1

Tudo

r TD

RD

1 2

Tudo

r TD

RD

1 4

Tudo

r TD

RD

1 3

Tudo

r TD

RD

6 6

Tudo

r EN

SG19

8639

3

Tudo

r TD

RD

9 1

Tudo

r AK

AP1

1

Tudo

r TD

RD

6 3

Tudor ENSG

198639 2Tudor TD

RD

6 2Tudor TD

RD

5 1Tudor R

NF17 1

Tudor TDRD6 1

Tudor TDRD6 7

Tudor TDRD10 1Tudor RNF17 2

Tudor ENSG198639 5

Tudor ECAT8 2

Tudor ECAT8

Tudor TDRD7 1

Tudor TDRD7 3

Tudo

r TD

RD

6 8

Tudo

r MYH

3 1

PWW

P ZC

WPW

2 1

Tudo

r SET

DB1

1

Tudo

r BAH

CC1

1

Tudo

r TNR

C18

1

Tudo

r ARI

D4B

1

Tudo

r ARID

4A 1

Tudo

r JMJD

2B 1

Tudo

r JMJD

2A 1

Tudo

r JMJD

2C 1

Tudor

MTF2 1

Tudor

PHF1 1

Tudor PHF19 1

Tudor LBR 1

Tudor JMJD2B 2

Tudor JMJD2C 2

Tudor JMJD2A 2

Tudor Tudorlik

e PHF20L1 2

Tudor Tudorlik

e PHF20 2

Tudor ZGPAT 1

PWWP BRD1 1

PWWP ARPC4 1

PWWP BRPF3 1

PWWP NP 115958.2 1

PWWP NSD1 2

PWWP WHSC1 2

PWWP WHSC1L1 2

PWWP HDGFL1 1

PWWP PSIP1 1

PWWP NP 001001520.1 1PWWP HDGR3 1

PWWP HDGF 1

PWWP DNMT3B 1PWWP DNMT3A 1PWWP PWWP2 1PWWP MBD5 1

PWWP MSH6 1PWWP WHSC1 1

PWWP WHSC1L1 1

PWWP NSD1 1

PWWP ZMYND11 1PWWP ZMYND8 1

PWWP MUM1 1

PWWP MUM1L1 1PWWP ZCWPW1 1

Mbt SFMBT1 3

Mbt SFMBT2 3

Mbt L3MBTL2 3

Mbt MBTD1 2

Mbt SCM

H1 2

Mbt SC

ML2 2

Mbt L3M

BTL4 3

Mbt L3M

BTL3 3

Mbt L3M

BTL 3

Mbt M

BTD1 4

Mbt L3M

BTL2 4

Mbt L3M

BTL 2

Mbt L3M

BTL3 2

Mbt L3M

BTL4 2

Mbt SCM

L2 1

Mbt SCM

H1 1

Mbt L3M

BTL3 1

Mbt L3M

BTL 1M

bt L3MBTL4 1

Mbt SFMBT1 4

Mbt SFM

BT2 4

Mbt SFMBT1 1Mbt SFMBT2 1

Mbt MBTD1 1Mbt L3MBTL2 1

Mbt SFMBT2 2

Mbt SFMBT1 2

Mbt L3MBTL2 2

Mbt MBTD1 3

Chrom

o SM

AR

CC

1 1Chr

omo

SM

AR

CC

2 1

Chr

omo

CH

D4

1

Chr

omo

CH

D5

2

Chr

omo

CH

D3

1

Chr

omo

MSL

3L1

1

Tudo

r PH

F20

1

Tudo

r Tud

orlik

e P

HF2

0L1

1

Mbt

PH

F20L

1 1

Chr

omo

ARID

4A 1

Tudo

r Chr

omo

AR

ID4B

2

Chr

omo

HTA

TIP

1

Chro

mo

MYS

T1 1

Agen

et F

MR1

1

Agen

et F

XR1

1

Tudo

rlike

Age

net

FXR2

1

Chromo CHD7 1

Chromo CHD9 1

Chromo CHD8 2

Chromo C

HD6 2

Chromo C

HD2 1

Chromo C

HD1 1

Chromo SUV39H2 1Chromo SUV39H1 1Chromo CBX2 1Chromo CBX7 1

Chromo CBX4 1Chromo CBX6 1Chromo CBX8 1

Chromo MPP8 1

Chromo CDYL2 1

Chromo CDYL 1

Chromo CDY2 1

Chromo CDY1 1

Chromo CBX1 1

Chromo CBX5 1

Chromo ENSG177447 1

Chromo CBX3 1Chromo LOC642721 1

Chromo CHD7 2Chromo CHD6 1Chromo CHD9 2

Chromo CHD8 1

Chromo CHD3 2Chromo CHD5 1

Chromo CHD4 2

Chromo CHD2 2

Chromo CHD1 2

Chro

mo

shad

ow C

BX5

2

Chrom

o sh

adow

CBX

1 2

Chrom

o sha

dow C

BX3 2

Chrom

o sha

dow E

NSG1774

47 2

shad

owLO

C642

721

1

shad

owEN

SG18

5790

1

A

'Royal family' domains

RRM


chromo domain: MBT domain:

PWWP domain:

Tudor domain:(Smn,SND1,germline)

ChSh

ECH

ANK

PTXAT_hook

SET

TCHSANTDEXD HELIC

C2H2DUFDEXD HELICCTNT

DEXD HELIC

KH KH

MOS-SAS

SWIRM SANT

C2H2 SAM-pnt

C2H2

SAM-pntSAM-pnt

SAM-pnt

PHD AWS SETHMG AWS SET

MusT I II III IV MusT CBromoPHD zfMYND

MBD

PHD DNA_methylase

BromoPHD

zfCW

Tudor domain (histone):

C2H2 PHD

RBP Bright Chromo

zfC3H1 Gpatch

jmjN PHDjmjC

ERG4_ERG24 DUF

PHD PHD

PHD

MBD SET

PHDMyosinN MYScIQ _tail

RBB BrightTudor

zfMYND

KHKHKH

HA2HELIC

RINGUBASNc SNc SNc SNc

PkinaseRRM

DEXDBRCT BRCT

B

PHD

BRKPHD RING

PHD

PHD

DEXD HELIC BRKSANTPHD

JMJD

Smn

SND1

fig. S10. An overview of the Royal Family of domains

A. A wheel constructed on results from multiple alignments of sequences of Chromo,

Chromo shadow, Agenet, Mbt, PWWP and Tudor domains (the degree of similarity is

represented by the hierarchical tree at the center). These domains likely derived from a

common ancestor (12). The domains are labeled with the domain name, followed by the

protein name, and finally the sequential order of the domain from N- to C-terminus in the

case of proteins with tandem repeats of the same domain type. The color bars at the

outer-most rim of the wheel show one or more of 60 co-existing domains (see legend to

the left) that are linked to a Royal family domain.

B. Domain architectures of proteins in A (color matched to legend in A).


Ago1 ---------------------------------------------------MEAGPS----------GAAAGAYLPPLQQVFQAPRRPGIG---------------------------------------------------------TVGKPIKLLANYFEVDIPK---------IDVY 53 Ago3 ---------------------------------------------------MEIGSA----------GPIG------AQPLFIVPRRPGYG---------------------------------------------------------TMGKPIKLLANCFQVEIPK---------IDVY 47 Ago4 ---------------------------------------------------MEALGP----------GPPA--------SLFQPPRRPGLG---------------------------------------------------------TVGKPIRLLANHFQVQIPK---------IDVY 45 Ago2 ---------------------------------------------------MYSGAGPVL-------ASPAPTTSPIPGYAFKPPPRPDFG---------------------------------------------------------TTGRTIKLQANFFEMDIPK---------IDIY 56 Miwi ----------------------------MT-------GRARARARGRARG----QETVQH------VGAAASQQPGYIPPR---PQQSPTE---GDLVGRGR-----QRG-------------------MVVGAT-------------SKSQELQISAGFQELSLAE---RGGR-RRDFH 88 Miwi2 -----------------MRLRILGVHRALP-------THARVAVCSNYLGKLEYSQTPSH------SHTVSFAKEKTLLLRLTSPGKPLAP---RNMSGRAR-----VRARGITTGHSAREVGRSSRDLMVTSASPGDSEAGGGTSVISQPYELGVSSGDGGRTFME---RRGKGRQDFE 139 Mili MDPVRPLFRGPTPVHPSQCVRMPGCWPQAPRPLEPAWGRAGPAGRGLVFRKPEDSSPPLQPVQKDSVGLVSMFRGMGLDTAFRPPSKREVPPLGRGVLGRGLSANMVRKDREEPRSSLPDPSVLAAGDSKLAEASVGWSRMLGRGSSEVSLLPLGRAASSIGRGMDKPPSAFGLTARDPP 180

. * : : :. : : * Ago1 HYE-----------------VDIKPDKCPRRVNREVVEYMVQHFKPQIFGDRKPVYDGKENIYTVTALPIGNERVDFEVTIPGEG---KDRIFKVSIKWLAIVSWRMLHEALVSGQIPVPLES---------------VQALDVAMR-HLASMRYTPVGRSFFSPPEGYYHPLGGGREVW 197 Ago3 LYE-----------------VDIKPDKCPRRVNREVVDSKVQHFKVTIFGDRRPVYDGKRSLYTANPLPVATTGVDLDVTLPGEGG--KDRPFKVSVKFVSRVSWHLLHEALAGGTLPEPLELDKPVS-------TNPVHAVDVVLR-HLPSMKYTPVGRSFFSAPEGYDHPLGGGREVW 200 Ago4 HYD-----------------VDIKPEKRPRRVNREVVDTMVRHFKMQIFGDRQPGYDGKRNMYTAHPLPIGRDRIDMEVTLPGEG---KDQTFKVSVQWVSVASLQLLLEALAGHLNEVPDDS---------------VQALDVITR-HLPSMRYTPVGRSFFSPPEGYYHPLGGGREVW 189 Ago2 HYE-----------------LDIKPEKRPRRVNREIVEHMVQHFKTQIFGDRKPVFDGRKNLYTAMPLPIGRDKVELEVTLPGEG---KDRILKVSIKWVSCVSLQALHDALSGRLPSVPFET---------------IQALDVVMR-HLPSMRYTPVGRSFFTASEGCSNPLGGGREVW 200 Miwi DLG-----------------VNTRQNLDHVKESKTGSSGIIVKLSTNHFRLTSRPQWALYQYHIDYNPLMEARRLRSALLFQHEDLIGRCHAFDGTILFLPKRLQHKVTEVFSQTRNGEHVRITITLTNELPPTSPTCLQFYNIIFRRLLKIMNLQQIGRNYYNPSDPIDIPNHR-LVIW 250 Miwi2 ELG-----------------VCTREKLTHVKDCKTGSSGIPVRLVTNLFNLDLPQDWQLYQYHVTYSPDLASRRLRIALLYNHSILSDKAKAFDGASLFLSEKLDQKVTELTSETQRGETIKITLTLTSKLFPNSPVCIQFFNVIFRKILKNLSMYQIGRNFYKPSEPVEIPQY------ 296 Mili RLPQPPALSPTSLHSADPPPVLTMERKEKELLVKQGSKGTPQSLGLNLIKIQCHN-EAVYQYHVTFSPSVECKSMRFGMLKDHQSVTGNVTAFDGSILYLPVKLQQVVELKSQRKTDDAEISIKIQLTKILEPCSDLCIPFYNVVFRRVMKLLDMKLVGRNFYDPTSAMVLQQHR-LQIW 358 : : . : : . : : : : . . :. : ::. : : : :: * : : :**.:: ... Ago1 FGFHQSVRPAMWKMMLNIDVSATAFYKAQPVIEFMCEVLDIRNIDEQPKPLTDSQRVRFTKEIKGLKVEVTHCGQMKRKYRVCNVTRRPASHQTFPLQLESGQTVECTVAQHFKQKYNLQLKYPHLPCLQVGQ---------EQKHTYLPLEVCNIVAGQRCIKKLTDNQTSTMIKATAR 368 Ago3 FGFHQSVRPAMWKMMLNIDVSATAFYKAQPVIQFMCEVLDIHNIDEQPRPLTDSHRVKFTKEIKGLKVEVTHCGTMRRKYRVCNVTRRPASHQTFPLQLENGQTVERTVAQYFREKYTLQLKYPHLPCLQVGQ---------EQKHTYLPLEVCNIVAGQRCIKKLTDNQTSTMIKATAR 371 Ago4 FGFHQSVRPAMWNMMLNIDVSATAFYRAQPIIEFMCEVLDIQNINEQTKPLTDSQRVKFTKEIRGLKVEVTHCGQMKRKYRVCNVTRRPASHQTFPLQLENGQAMECTVAQYFKQKYSLQLKHPHLPCLQVGQ---------EQKHTYLPLEVCNIVAGQRCIKKLTDNQTSTMIKATAR 360 Ago2 FGFHQSVRPSLWKMMLNIDVSATAFYKAQPVIEFVCEVLDFKSIEEQQKPLTDSQRVKFTKEIKGLKVEITHCGQMKRKYRVCNVTRRPASHQTFPLQQESGQTVECTVAQYFKDRHKLVLRYPHLPCLQVGQ---------EQKHTYLPLEVCNIVAGQRCIKKLTDNQTSTMIRATAR 371 Miwi PGFTTSILQYENNIMLCTDVSHKVLRSET-VLDFMFNLYQQTEEHKFQEQVS--------KELIGLIVLTKYN---NKTYRVDDIDWDQNPKSTFKKA----DGSEVSFLEYYRKQYNQEITDLKQPVLVSQP-KRRRGPGGTLPGPAMLIPELCYLTGLTDKMRNDFNVMKDLAVHTRL 413 Miwi2 -----------NKLLFNADVNYKVLRNET-VLDFMTDLCLRTGMSCFTEMCH--------KQLVGLVVLTRYN---NKTYRIDDIDWSVKPTQAFQKR----DGSEVTYVDYYKQQYDITLSDLNQPVLVSLL-KRKRN-DNSEPQMVHLMPELCFLTGLSSQATSDFRLMKAVAEETRL 447 Mili PGYAASIRRTDGGLFLLADVSHKVIRNDS-VLDVMHAIYQQNKE-HFQDECS--------KLLVGSIVITRYN---NRTYRIDDVDWNKTPKDSFVMS----DGKEITFLEYYSKNYGITVKEDDQPLLIHRPSERQNNHGMLLKGEILLLPELSFMTGIPEKMKKDFRAMKDLTQQINL 512 ::: **. ..: :::.: * : * * : .:.**: :: . .:* : * : ::: ..: : . * * : ::* . . : Ago1 SAPDRQEEISRLMKNASCN--LDPYIQ-----------EFGIK---VKDDMTEVTGRVLPAPILQYGGRNRAIATP-----------NQGVWDMRGKQFYNGIEIKVWAIACFAPQKQCREEVLKNFTDQLRKISKDAGMPIQGQPCFCKYAQGADSVEPMFRHLKNTYSGLQLIIVILP 521 Ago3 SAPDRQEEISRLVRSANYE--TDPFVQ-----------EFQLK---VRDEMAHVTGRVLPAPMLQYGGRNRTVATP-----------SHGVWDMRGKQFHTGVEIKMWAIACFATQRQCREEILKGFTDQLRKISKDAGMPIQGQPCFCKYAQGADSVEPMFRHLKNTYSGLQLIIVILP 524 Ago4 SAPDRQEEISRLVKSNSMVGGPDPYLK-----------EFGIV---VHNEMTELTGRVLPAPMLQYGGRNKTVATP-----------SQGVWDMRGKQFYAGIEIKVWAVACFAPQKQCREDLLKSFTDQLRKISKDAGMPIQGQPCFCKYAQGADSVEPMFKHLKMTYVGLQLIVVILP 515 Ago2 SAPDRQEEISKLMRSASFN--TDPYVR-----------EFGIM---VKDEMTDVTGRVLQPPSILYGGRNKAIATP-----------VQGVWDMRNKQFHTGIEIKVWAIACFAPQRQCTEVHLKSFTEQLRKISRDAGMPIQGQPCFCKYAQGADSVEPMFRHLKNTYAGLQLVVVILP 524 Miwi TPEQRQREVGRLIDYIHKDDNVQRELR-----------DWGLS---FDSNLLSFSGRILQSEKIHQGGKTFDYNP----------QFADWSKETRGAPLISVKPLDNWLLIYTRR----NYEAANSLIQNLFKVTPAMGIQMKKAIMIEV-DDRTEAYLRALQQKV--TSDTQIVVCLLS 562 Miwi2 SPVGRQQQLARLVDDIQRTLPSSQEVLSHTSLPLWAPEPGGLSSAIPLSTVLPFAQQLLTALSLSPGIPLPHLKPPSFLFLCQPAFAADWSKDMRSCKVLSSQPLNRWLIVCCNR----AEHLIEAFLSCLRRVGGSMGFNVGYPKIIKV-DETPAAFLRAIQVHG--DPDVQLVMCILP 620 Mili SPKQHHGALECLLQRISQNETASNELT-----------RWGLS---LHKDVHKIEGRLLPMERINLRNTSFVTSED-----------LNWVKEVTRDASILTIPMHFWALFYPKR----AMDQARELVNMLEKIAGPIGMRISPPAWVELKDDRIETYIRTIQSLLGVEGKIQMVVCIIM 672 :. :: : *: . : : . : . ::* : . : :. * : . : . * :: *: : . : : :: *::: :: Ago1 G-KTPVYAEVKRVGDTLLGMATQCVQVKNAVK--TSPQTLSNLCLKINVKLGGINNILVPHQRSAVFQQPVIFLGADVTHPPAGDGKKPSITAVVGSMDAHPSRYCATVRVQRPRQ----------EIIEDLSYMVRELLIQFYKSTRFKPTRIIFYRDGVPEGQLPQILHYELLAIRDA 688 Ago3 G-KTPVYAEVKRVGDTLLGMATQCVQVKNVIK--TSPQTLSNLCLKINVKLGGINNILVPHQRPSVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPSRYCATVRVQRPRQ----------EIIQDLASMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFRQVLYYELLAIREA 691 Ago4 G-KTPVYAEVKRVGDTLLGMATQCVQIKNVVK--TSPQTLSNLCLKMNAKLGGINNVPVPHQRPSVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDGHPSRYCATVWVQTSRQEIAQELLYSQEVVQDLTSMARELLIQFYKSTRFKPTRIIYYRGGVSEGQMKQVAWPELIAIRKA 692 Ago2 G-KTPVYAEVKRVGDTVLGMATQCVQMKNVQR--TTPQTLSNLCLKINVKLGGVNNILLPQGRPPVFQQPVIFLGADVTHPPAGDGKKPSIAAVVGSMDAHPNRYCATVRVQQHRQ----------EIIQDLAAMVRELLIQFYKSTRFKPTRIIFYRDGVSEGQFQQVLHHELLAIREA 691 Miwi SNRKDKYDAIKKYLCTDCPTPSQCVVARTLGKQQTVMAIATKIALQMNCKMGG------ELWRVDMPLKLAMIVGIDCYHDTTA--GRRSIAGFVASINEGMTRWFSRCVFQDRGQ----------ELVDGLKVCLQAALRAWSGCNEYMPSRVIVYRDGVGDGQLKTLVNYEVPQFLDC 724 Miwi2 SNQKNYYDSIKKYLSSDCPVPSQCVLTRTLNKQGTMLSVATKIAMQMTCKLGG------ELWSVEIPLKSLMVVGIDICRDALN--KNVVVVGFVASINSRITRWFSRCVLQRTAA----------DIADCLKVCMTGALNRWYRHNHDLPARIVVYRDGVGNGQLKAVLEYEVPQLLKS 782 Mili GTRDDLYGAIKKLCCVQSPVPSQVINVRTIGQPTRLRSVAQKILLQMNCKLGG------ELWGVDIPLKQLMVIGMDVYHDPSR--GMRSVVGFVASINLTLTKWYSRVVFQMPHQ----------EIVDSLKLCLVGSLKKYYEVNHCLPEKIVVYRDGVSDGQLKTVANYEIPQLQKC 834 . : * :*: .:* : :. : :: :::. *:** : : :.:* * : . :...*.*:: .:: : .* :: : * * : .. * ::: **.** :**: : *: : .. Ago1 CIKLEKDYQPGITYIVVQKRHHTRLFCADKNERIGKSGNIPAGTTVDTNITHPFEFDFYLCSHAGIQGTSRPSHYYVLWDDNRFTADELQILTYQLCHTYVRCTRSVSIPAPAYYARLVAFRARYHLVDKEHDSGEGSHISGQSNGRDPQALAKAVQVHQDTLRTMYFA 857 Ago3 CISLEKDYQPGITYIVVQKRHHTRLFCADRTERVGRSGNIPAGTTVDTDITHPYEFDFYLCSHAGIQGTSRPSHYHVLWDDNFFTADELQLLTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHVSGQSNGRDPQALAKAVQIHQDTLRTMYFA 860 Ago4 CISLEEDYRPGITYIVVQKRHHTRLFCADKMERVGKSGNVPAGTTVDSTVTHPSEFDFYLCSHAGIQGTSRPSHYQVLWDDNCFTADELQLLTYQLCHTYVRCTRSVSIPAPAYYARLVAFRARYHLVDKDHDSAEGSHVSGQSNGRDPQALAKAVQIHHDTQHTMYFA 861 Ago2 CIKLEKDYQPGITFIVVQKRHHTRLFCTDKNERVGKSGNIPAGTTVDTKITHPTEFDFYLCSHAGIQGTSRPSHYHVLWDDNRFSSDELQILTYQLCHTYVRCTRSVSIPAPAYYAHLVAFRARYHLVDKEHDSAEGSHTSGQSNGRDHQALAKAVQVHQDTLRTMYFA 860 Miwi LKSVGRGYNPRLTVIVVKKRVNARFFAQSG----GRLQNPLPGTVIDVEVTRPEWYDFFIVSQAVRSGSVSPTHYNVIYDSSGLKPDHIQRLTYKLCHVYYNWPGVIRVPAPCQYAHKLAF------------------LVGQSIHREP---------NLSLSNRLYYL 862 Miwi2 VTECG----------------------------------------------SDARYDFYLISQTANRGTVSPTHYNVIYDDNALKPDHMQRLTFKLCHLYYNWQGLISVPAPCQYAHKLTF------------------LVAQSVHKEP---------SLELANNLFYL 878 Mili FEAFDN-YHPKMVVFVVQKKISTNLYLAAP----DHFVTPSPGTVVDHTITSCEWVDFYLLAHHVRQGCGIPTHYICVLNTANLSPDHMQRLTFKLCHMYWNWPGTIRVPAPCKYAHKLAF------------------LSGQILHHEP---------AIQLCGNLFFL 971 **:: :: * *:** : : :..*.:* **::*** * . : :***. **: :: * .* :: . :::

fig. S11. Sequence characteristics of the mouse Piwi clade of the Argonaute proteins

Sequence alignment of Argonaute family proteins was conducted using ClustalW

programming (www.ebi.ac.uk/clustalw/). Note that Piwi clade and Ago clade proteins

differ at their N-termini, where they show a low degree of overall sequence similarity as

compared to the rest of the sequences, which are more highly conserved (compare the

frequency of conserved residues, indicated below the alignment). Of interest, The Piwi

clade proteins have multiple arginine-glycine motifs (RG-boxes – highlighted) in their N-

termini, which are absent from the Ago proteins.

Q- (2me)R53 -G -M-V-V-G -A-T-S-K

K S T A G VV M+o G R+2me QKSTAGV VM+oGR+2meQ

fig. S12. An MS/MS spectrum of a tryptic peptide containing dimethylated (2me) Arg53 of endogenous Miwi protein from testis.


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Supplementary Materials for - Linding Lab...Rich1 and Tuba localize to cell-cell junctions, associate with proteins involved in junction formation and cell polarity, and are active