cloning of human abba-1 - the journal of biological … · cloning of human abba-1—hum an abba- 1...

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SUPPLEMENTAL DATA Collection of nucleotide sequence data of putative members of the IRSp53/MIM-family proteins. Cloning of human ABBA-1—Human ABBA-1 (F-a ctin-b undling protein with B AIA P2 homology in the N-terminal helical region, AB115770) was cloned from a human brain cDNA library (pDNR-LIB, Clontech) by PCR amplification using a pair of primers predicted from the human genomic sequence. The primers used were 5’AGAGCATGGAGACGGCGGAGAAGGAGTG (forward) and 5’CACCTGCTCGCACTGGGGCCTGAGAG (reverse) found in a hypothetical gene located at 16q22.1 (LocusID: 92154). The model mRNA of this gene, XM_208937, encodes a polypeptide similar to the C-terminal half of MIM. There are putative exons encoding amino acid sequences highly homologous to the N-terminal half of MIM in the upstream region. Expression of these putative exons is supported by expression sequence tags (ESTs) such as AL539489 and BU859607. The 2294 bp PCR product obtained has an open reading frame of 747 amino acids that shows 55% identity and 11% similarity to human MIM. The coding region of human ABBA-1 spans 22.2 kb and consists of 15 exons on the minus strand of a genomic contig NT_010498.13: join (19441131..19441197, 19436376..19436431, 19436201..19436273, 1

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Page 1: Cloning of human ABBA-1 - The Journal of Biological … · Cloning of human ABBA-1—Hum an ABBA- 1 ... human ABBA-1 showed a single 6 kb band in various tissues with the highest

SUPPLEMENTAL DATA

Collection of nucleotide sequence data of putative members of the IRSp53/MIM-family

proteins.

Cloning of human ABBA-1—Human ABBA-1 (F-actin-bundling protein with BAIAP2

homology in the N-terminal helical region, AB115770) was cloned from a human brain cDNA

library (pDNR-LIB, Clontech) by PCR amplification using a pair of primers predicted from the

human genomic sequence. The primers used were

5’AGAGCATGGAGACGGCGGAGAAGGAGTG (forward) and

5’CACCTGCTCGCACTGGGGCCTGAGAG (reverse) found in a hypothetical gene located at

16q22.1 (LocusID: 92154). The model mRNA of this gene, XM_208937, encodes a polypeptide

similar to the C-terminal half of MIM. There are putative exons encoding amino acid sequences

highly homologous to the N-terminal half of MIM in the upstream region. Expression of these

putative exons is supported by expression sequence tags (ESTs) such as AL539489 and

BU859607. The 2294 bp PCR product obtained has an open reading frame of 747 amino acids

that shows 55% identity and 11% similarity to human MIM. The coding region of human

ABBA-1 spans 22.2 kb and consists of 15 exons on the minus strand of a genomic contig

NT_010498.13: join (19441131..19441197, 19436376..19436431, 19436201..19436273,

1

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19435377..19435461, 19435192..19435283, 19435036..19435110, 19434720..19434728,

19433658..19433815, 19433198..19433305, 19429712..19429934, 19420872..19420946,

19420326..19420502, 19420004..19420169, 19419038..19419855). All the exons except one

keep AG/exon/GT boundaries and the exon 13 uses GC instead of GT. Northern blot analysis of

human ABBA-1 showed a single 6 kb band in various tissues with the highest expression level in

the brain.

Cloning of Zebrafish IRSp53—Zebrafish IRSp53 was PCR-amplified from Zebrafish adult brain

cDNAs using a pair of primers predicted from the zebrafish cDNAs and the genomic sequences

(An UniGene cluster Dr.16661, WGS 126238745 and a genomic contig BX323027). The forward

primer was 5’TCTCCTCCTCCTCCTCTTGTGTTTTCTCG and the reverse primer was

5’AGCATGTCTGATAAATGTGTATGAAAGGTG. The 1771 bp PCR product obtained has an

open reading frame of 534 amino acids that shows 63% identity and 17% similarity to human

IRSp53. The brain cDNAs were kindly gifted from Dr. M. Hibi (Riken Center for Developmental

Biology).

Cloning of Zebrafish ABBA-1 and -2—Zebrafish ABBA-1 and -2 were similarly cloned from the

brain cDNAs using two pairs of PCR primers predicted from the zebrafish database. We found

two N-terminal and two C-terminal sequences that are more closely related to ABBA-1 than

MIM. A forward primer (F1) 5’TTCTTCCAGCCGATTTTTGTTCATTCATTGGGAGCAG was

2

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predicted from ESTs AL914911 and AL914912 and the other one (F2)

5’TTGGGCTGGTAAAATGGATGCGGGAATGG was predicted from genomic sequences

BX530017 and BX548167. Similar to human ABBA-1, the last exons of zebrafish putative

ABBAs encode one third of the whole proteins, so that we could obtain two different relatively

long 3’-sequences from AI959191 and overlapping WGSs and from BQ419430 and WGSs. The

predicted 3’ primers were R1: 5’TGAAAAGCTTATTTAGTGTGATCGGGGCCTTAGACG and

R2: 5’AGGAGGCATCACAAAATCCGGGGTGCAGAG. Only the combinations of F1/R1 and

F2/R2 successfully amplified PCR-products of 2240 bp and 2222 bp, respectively, and were

tentatively named ABBA-1 and ABBA-2. The deduced aa sequence of zebrafish ABBA-1

contains 710 aa and shows 66% identity/9% similarity to human ABBA-1 and 51% identity/12%

similarity to human MIM. ABBA-2 contains 737 aa that shows 73% identity/10% similarity to

human ABBA-1 and 56% identity/11% similarity to human MIM.

Database searches for putative IRSp53/MIM family proteins in vertebrates—We found five

candidates proteins in the human genome, IRSp53, IRTKS, FLJ22582, MIM and ABBA-1. To

reveal the conservation of these proteins in vertebrates, we intensively searched Gallus gallus

(chicken) sequences for the bird, Xenopus laevis (african clawed frog) and Silurana tropicalis

(western clawed frog) sequences for the amphibia, and Danio reio (zebra fish) sequences for the

3

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fish. Basically overlapping EST clones were used to make putative full-length mRNA sequences.

Mismatches and possible sequence errors were corrected by overlapping ESTs and genomic

sequences, mainly whole genome shotgun (WGS) sequences. We have found that the exon

compositions of these genes are almost completely conserved and more than five WGSs could be

obtained for most of the exons. GenBank accession numbers of representative clones used are

sited below.

IRSp53 sub-family

Human IRSp53 isoform 1. NP_059344.

Mouse IRSp53. NP_570932.

Bovine IRSp53. UniGene Bt.5610; ESTs, BF654505, BI847652, AV602077, BF604061,

BF040137, AV612697, BI541686, BE721861.

Chicken IRSp53. BM440642, BG712911, CD218196, BM491233, BI847652.

Silurana tropicalis IRSp53. UniGene Str.6299; the N-terminal fragments, AL892953, AL847406.

The C-terminal fragment, AL864224.

Human IRTKS. NP_061330.

Mouse IRTKS. NP_080109.

Bovine IRTKS. the C-terminal fragment, UniGene Bt.11916; BF044090, BF046149, BF045001,

CB437083.

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Chicken IRTKS. BU473774, BU129548, BX262287, BU293404.

Zebra fish IRTKS. AAH50238, UniGene Dr.11420.

Human FLJ22582. NP_079321.

Mouse FLJ22582. AAH48937.

Chicken FLJ22582. CD728910, BU307330, CD733604, BU372164, BU305484.

Xenopus FLJ22582. The C-terminal fragment, UniGene Xl.19687, CA972469, CA973430,

BQ383816, BQ383815.

Zebra fish FLJ22582. The N-terminal fragment, AL916895, CA974969, CD595810, BI980054.

MIM sub-family

Human MIM. NP_055566.

Mouse MIM. NP_659049.

Bovine MIM. The C-terminal fragment, AW437626, BF043708, BF076606, BE476284.

chicken MIM. BU446345, BU359736, BU359378, BU131818, BI066132, BM426009,

BU257939.

Xenopus MIM. The N-terminal fragment, BU913061, BU913016, BQ726165. The C-terminal

fragment, BU916773, BI444844.

Mouse ABBA-1. Partial sequence was found in the locus 244654 on chromosome 8, whereas

mouse MIM is encoded by the locus 211401 on chromosome 15. The model sequence,

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XM_146515, was extended and edited by a number of ESTs such as CB525721, CD355768,

CD348498 and AK052172. There may be splice isoforms. The sequence shown is the closest

form to our human ABBA-1 clone. There is a rat ortholog, XM_226503. chicken ABBA-1. The

N-terminal fragment, BU372800, BU142478, BU396053, BU372986.

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Hs IRSp53 1 MSLSRSEEMHRLTENVYKTIMEQFNPSLRNFIAMGKNYEKALAGVTYAAKGYFDALVKMGELASESQGSKMm IRSp53 1 MSLSRSEEMHRLTENVYKTIMEQFNPSLRNFIAMGKNYEKALAGVTFAAKGYFDALVKMGELASESQGSKBt IRSp53 1 MSLSRSEEMHRLTENVYKTIMEQFNPSLRNFIAMGKNYEKALAGVTYAAKGYFDALVKMGELASESQGSKGg IRSp53 1 MARSEEVHRLTENVYKTIMEQFNPSLRNFISMGKTYEKALASMTYAAKGYFDALVKMGELASESQGSKSt IRSp53-N 1 MSRSDEVNRLTENVYKTIMEQFNPCLRNFIAMGKNYEKALASVTFAAKGYFDALVKMGELASESQGSKDr IRSp53 1 MSRTDEVHRITENVYKSIMEQFNPCLRNFVAMGKTYEKALSSVTFAAKGYFDALVRMGELASESHGSK ..*..*..*.******.*******.****..***.*****. .*.**********.********.***

Hs IRSp53 71 ELG----DVLFQMAEVHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTEQRSKGDALDKMm IRSp53 71 ELG----DVLFQMAEVHRQIQNQLEETLKSFHNELLTQLEQKVELDSRYLSAALKKYQTEQRSKGDALDKBt IRSp53 71 ELG----DVLFQMAEVHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTEQRSKGDALDKGg IRSp53 69 ELG----DVLFQMAEVHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTEQRNKGDSLDKSt IRSp53-N 69 ELG----DVLFQMAEVHRQIQNQLEEMLKSLHNELLTQLEQKVELDSRYLNAALKKYQVEHKNKGEALEKDr IRSp53 69 DLEAAWEDVLFQMAEVHRQIQMQLEDMLKSFHNELLTELEKKVELDVRYLNAALKKYQMEHKSKGESLEK .* ************** ***. *** ******.**.***** ***.******* *...**..*.*

Hs IRSp53 137 CQAELKKLRKKSQGSKNPQKYSDKELQYIDAISNKQGELENYVSDGYKTALTEERRRFCFLVEKQCAVAKMm IRSp53 137 CQAELKKLRKKSQGSKNPQKYSDKELQYIDAISNKQGELENYVSDGYKTALTEERRRFCFLVEKQCAVAKBt IRSp53 137 CQAELKKLRKKSQGSEGRQKYSDKELQYIDAIGNKQGELESYVSDGYKTALTEERRRFCFLVEKQCAVAKGg IRSp53 135 CQAELKKLRKKSQGSKNPQKYSDKELQYIEAISNKQGELENYVSDGYKTALTEERRRFCFLVEKQCAVAKSt IRSp53-N 135 CQVELKKLRKKSQGSKNPQKYSDKEMQYIEAISNKQGELESHISDGYKTALTEERRRYCFLVEKQCA-201Dr IRSp53 139 CQAELKKLRRKSQGSKNPSKYGDKEMQYVETISSKQNELDNYISESYKNALSEERRRYCFLVDRQCAVAK ** ************ .** ***.**.. * .** **.. .*. **.**.*****.****..******

Hs IRSp53 207 NSAA-YHSKGKELLAQKLPLWQQACADPSKIPERAVQLMQQVA--SNGATLPSAL--SASKSNLVISDPIMm IRSp53 207 NSAA-YHSKGKELLAQKLPLWQQACADPNKIPDRAVQLMQQMA-NSNGSILPSAL--SASKSNLVISDPIBt IRSp53 207 NSAG-YHSKGKELLAQKLPLWQQACADPNKIPDRAVQLMQQMG-NNNGVILPRAL--SASKSNLVISDPIGg IRSp53 205 -SAITYHAKGKEMLTQKLPLWQQSCSDPNKIPDRAIQLMQQMAASSNGTIMPSPL--STSKSNLIISDPIDr IRSp53 209 TSN-TYHTKGRDLLTQKIPLWQQACADPNKLPERAMLLAQQMSSGAAGTLGPSV-HHT-SKSNLTISDPI .* ** **...* **.*****.****.*.*.**. * **. * * . . ***** *****

Hs IRSp53 272 PGAKPLPVPPELAPFVG----RMSAQESTPIMNGVTGPDGEDYSPWADRKAAQPKSLSPPQSQSKLSDSYMm IRSp53 273 PGAKPLPVPPELAPFVG----RMSAQENVPVMNGVAGPDSEDYNPWADRKAAQPKSLSPPQSQSKLSDSYBt IRSp53 273 PRAKPLPVPPELAPFVG----RLSAQENAPVMNGVSGPDSEDYNPWADRKATQPKSTSPPQSQSKLSDSYGg IRSp53 272 PGAKPLPVPPELAPFVG----RM--QEAMPAVNGVSGPDSDDYNHWSEVKPAQPKSLSPPQPQKQLSDSYSt IRSp53-C 1 ------------------------------------------------------ IPREK---SDSYDr IRSp53 276 PGAKPLPVPPELAALMGNQQSRMMGADSVPIVNGSGVHVGEDYQRWMETRAAHDKQSQQPHRHGGESFSN * *********** .* *. . * .** .** * . . . *. . *. * *

Hs IRSp53 338 SNTLPVRKSVTPKNSYATTENKTLPRSSSMAAGLERNGRMRVKAIFSHAAGDNSTLLSFKEGDLITLLVPMm IRSp53 339 SNTLPVRKSVTPKNSYATTENKTLPRSSSMAAGLERNGRMRVKAIFSHAAGDNSTLLSFKEGDLITLLVPBt IRSp53 339 SNTLPVRKSVAPKNSYATTENKTLPRSSSMAAGLERNGRMRVKAIFSHAAGDNSTLLSFKEGDLITLLVPGg IRSp53 336 SNTLPVRKNVTPKNSYASAENKTLPRSSSMAAGLERNGRTRVQAIFSHAAGDNSTLLSFKEGDLITLLVPSt IRSp53-C 11 SNTLPARKSVPPKNSYTVAENKTLPRSSSMAAGLERNGRTRVQAIFSHAAGDNTTLLSFKDGDYITLLVPDr IRSp53 346 TLPVRKVAPVKPKNTLA--ETQTLPRSSSMTTGLERNGRTRVQALFSHAAGDNSTLLSFTEGDIITLLVP . . * ***. . *..******** ******* **.*.********.***** .** ******

Hs IRSp53 408 EARDGWHYGESEKTKMRGWFPFSYTRVL-DSDGSDRLHMSLQQGKSSSTGNLLDKDDLAIPPPDYGAASRMm IRSp53 409 EARDGWHYGESEKTKMRGWFPFSYTRVL-DSDGSDRLHMSLQQGKSSSTGNLLDKDDLALPPPDYGTSSRBt IRSp53 409 EARDGWHYGESEKTKMRGWFPFSYTRVL-DNDGGDRLHMSLQQGKSSSTGNLLDKEDLALPPPDYGTSSRGg IRSp53 406 EARDGWHYGESEKTKLRGWFPFSYTRVL-DNDGSERVHASLQQGKSSSTGNLLDKDDIAIPPPDYGMSSQSt IRSp53-C 81 EARDGWHYGECEKTKLRGWFPFSYTRVL-DT-GTDRTQTSLQPGKSSSTGNLLDKDDLSIPPPDYNMASRDr IRSp53 414 EARDGWHYGENEKNRMRGWFPFSYTRLIAEPD-SNKLHVSLQHNRSSSTGNLLER-EMALPTPDYGMPTR ********** **...**********.. . .. . . *** .********.......* *** ..

Hs IRSp53 477 AFPAQTASG-FKQRPYSVAVPAFSQGLDDYGA-RSMSSGSGTLVSTV 521Mm IRSp53 478 AFPTQTAGT-FKQRPYSVAVPAFSQGLDDYGA-RSVSR 513Bt IRSp53 478 AFPTQTAGA-FKQRPYSVAVPAFSQGLDDYGA-RAVSRNPFASVQLKPTVTNDMVCPTS 534Gg IRSp53 475 AFPSQNVNT-FKQRPYSVAVPAFSQGLDDYGT-RSVSSGSSSLVSTV 519St IRSp53-C 149 GFQPP-GST-FKPRPYSVAAPVFSQGIEDYGAARPTNSGNGTMISTV 216Dr IRSp53 482 -FAQQQSTAHSRQRPYSVAVPGFSQV-SRYHT-HTLSTLSAAPLCTQRLTLTDICIY 535 * . ****** * *** . * . . .

Supplemental Fig. 1. Alignment of IRSp53 proteins.

The IMD is boxed. The half-CRIB motif, the SH3 domain and the WW binding motif are shown in a black box, shaded areas and a shaded box, respectively. Asterisks show the identity and dots show the similarity. Species: Hs, human; Mm, mouse; Bt, bovine; Gg, chicken; St, Silurana tropicalis; Dr, zebra fish. For detail, see Collection of nucleotide sequence data of putative members of the IRSp53/MIM family proteins.

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Hs IRTKS 1 MSRGPEEVNRLTESTYRNVMEQFNPGLRNLINLGKNYEKAVNAMILAGKAYYDGVAKIGEIATGSPVSTE Mm IRTKS 1 MSRGPEEVNRLTENTYRNVMEQFNPGLRNLINLGKNYEKAVNAMILAGKAYYDGVAKIGEIATGSPVSTE Gg IRTKS 1 MEQFNPGLRNLINLGKNYEKAVNAMVVAGRSYYDSLAKIGDISADSPVSKE Dr IRTKS 1 MSRASEDVNKLTESTYKNVMEQFNPGLRNLVNLGKNYEKSVAAMTFAGKAYFDALSKIGESAAVSPVSRE ... ****.... .....***********.********.* ******..*.* ..***. .. ****.*

Hs IRTKS 71 LGHVLIEISSTHKKLNESLDENFKKFHKEIIHELEKKIELDVKYMNATLKRYQTEHKNKLESLEKSQAEL Mm IRTKS 71 LGHVLIEISSTHKKLNETLDENFKKFHKDIIHELEKKTELDVKYMNATLKRYQAEHRNKLDSLEKSQAEL Gg IRTKS 52 LGQVLVEISRTHKKLNDSLEESFKKFHKEIISELEKKTDLDVKYMTATLKRYQTEHRSKLDSLEKSQAEL Dr IRTKS 71 LGVVLMEISEVHKNAQLEMEESFKRFHKELIAELEKKTDMDTKYMTATFKRYQSEHKLKQDFLDKSQADL ** **.***..**... ..* **.***..* ******..* *** ** ****.**. * . *.****.*

Hs IRTKS 141 KKIRRKSQGSRNALKYEHKEIEYVETVTSRQSEIQKFIADGCKEALLEEKRRFCFLVDKHCGFANHIHYY Mm IRTKS 141 KKIRRKSQGGRNALKYEHKEIEYVETVTSRQSEIQKFIADGCKEALLEEKRRFCFLVDKHCSFASHIHYY Bt IRTKS-C 1 KSQGGRNPFKYECKEIEYVETITSRQNEIQKFIADGCKEALLEEKRRFCFLVDKHCSFSSQIHHY Gg IRTKS 122 KKIRRKSQGARNVMKYEHKEMEYLETVSSRQTDIQRFIAEGCREALLEEKRRFCFLVDKHCSFTQHMHFYDr IRTKS 141 KKLRRKSQG-KHSSKYEIKENECMETISSRQTDMQRFIADGCKEALLEEKRRFCFLVDKHCALSYQLSAF **** .. *** ** * .**..*** ..*.***.**.****************** . .. .

Hs IRTKS 211 HLQSAELLNSKLPRWQETCVDAIKVPEKIMNMIEEIKTPASTPVSGTPQASPMIERSNVVRKDYDTLSKC Mm IRTKS 211 HMQSAELLNSKLPRWQETCCDATKVPEKIMNMIEEIKTPISTPVSGTPQPSPMIERSKMIGKDYDTLSKY Bt IRTKS-C 66 HMQSAELINSKLPRWQETCSDVTKVPEKIMNMIEEIKTPISTPMSGTPLPSPMIERSSMVGQDCDSLSKY Gg IRTKS 192 HAQCAEFLNAKLPGWQEICSDATKVPEKVKMMIEEIRTPGSTPISGTPQPSPMIERNTMIGNNFYAHNEN Dr IRTKS 210 HDKAKEMLTLKLPSWQEKCNDATRVPDTVISMIEGLRTSMSVIPESSP----TLERS-------DRTNSG * .. * .. *** *** * * .**. . *** ..* * .* .**

Hs IRTKS 281 SPKMPPAPSGRAYTSPLIDMFNNPATAAPNS-QRVNNSTGTS-EDPSLQRSVSVATGLNMMKKQKVKTIF Mm IRTKS 281 SPKMPPAPSVKAYTSPLIDMFNNPATAAQSS-EKTNNSTANTGEDPSLQRSVSVATGLNMMKKQKVKTIFBt IRTKS-C 136 PPKIPAAPSARALTSPLIDLFKNPATVAQNS-ERKNNLTGSS-VDPNLPRSVSMASGLNMVKKQKVKTIF Gg IRTKS 262 ASKVPPAPVGRAYTSPLVDMFNNPAMAPKVSPERPTNPAENS-DDASLSRSTSVATGLNIMKRQKVKTIFDr IRTKS 269 SIVPPPAPSLKAHTSPLASMFSQEAPKSPISSEQYSDQDSLD--ENGLSRSTSVSSGMDTAKKTRVQTIF * ** .* **** .* . * * .. . * ** *...*. *. .*.***

Hs IRTKS 349 PHTAGSNKTLLSFAQGDVITLLIPEEKDGWLYGEHDVSKARGWFPSSYTKLLEENET-EAVTVPTPSPTPMm IRTKS 350 PHTAGNNKTLLSFAQGDVLTLLIPEEKDGWLYGEHDTTKARGWFPSSYTKLLEEN---EAMSVPTPSPAPBt IRTKS-C 204 PHTAGANQTLLSFAQGDVITLLISEEKDGWMYGEHDSTKMRGWFPSSYTKLLEENEE-EAASELAPSLAP Gg IRTKS 331 PHTAGNNKTLLSFAQGDTIILLVSEEKDGWLYGEHESTKVKGWFPSSYTRPLEESE--EKAFVPASSAAP Dr IRTKS 337 PHTAGNNETLLSFEDGDIIILLIQEERDGWLYGELEHTGQRGWFPSSYCRSYNETTISNRMESPFSSHID ***** * ***** ** . **. **.***.*** . . .*******.. * *

Hs IRTKS 418 VRSISTVNLSENSSVVIPPPDYLECLSMGAAADRRADSART--TSTFKAPASKP--ETAAPNDANGTAKP Mm IRTKS 417 VRSISTVDLTEKSSVVIPPPDYLECLSMGATSDKKAGAPKVPSASTFRAPVSRPDATSTSPSDANGTAKPBt IRTKS-C 273 VRSVSTTNLAEKSSVVIPPPDYLEFLSTGAAAAEREDISQK--ASNHKAPVPKP--ESTPPNNANGFAKP Gg IRTKS 399 VRSISSVNLAEKAGVVLPPPDYLGSTSD-----KRMESSKG---TAARTPTDKPE-SAGLKPDMNGIVKP Dr IRTKS 407 LPEQQEEDEEVEEPVLLPPPDYSDSSRD------RPIKPST--------PSSQS-----TVSLANGHSMP . *..***** . * . . ** *

Hs IRTKS 484 PFLSGENPFATVKLRPTVTNDRSAPIIR 511Mm IRTKS 487 PFLSGENPFATVKLRPTVTNDRSAPIIR 514Bt IRTKS-C 339 PFLSGENPFATVKLRPTVTNDRSAPIIR 366Gg IRTKS 460 PFLSGENPFAAVKLRPTVTNDRSAPIIR 487Dr IRTKS 458 PFLGGGNPFATVRLRPTVTNDRSAPII 484 *** * ****.*.**************.

Supplemental Fig. 2. Alignment of IRTKS proteins.

The IMD is boxed. The SH3 domain is shaded and the WW binding motif is in a shaded box. Asterisks show the identity and dots show the similarity. Species: Hs, human; Mm, mouse; Bt, bovine; Gg, chicken; St, Silurana tropicalis; Dr, zebra fish. For detail, see Collection of nucleotide sequence data of putative members of the IRSp53/MIM family proteins.

8

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Hs FLJ22582 1 MAPEMDQFYRSTMAIYKSIMEQFNPALENLVYLGNNYLRAFHALSEAAEVYFS Mm FLJ22582 1 MAPEMDQFYRSTMAIYKSIMEQFNPALENLVYLGNNYLRAFHALSEAAEVYFSGg FLJ22582 1 MDLSYRSTISIYKSILEQFNPALENLVYLGNNYLRAFHALSKAAEVYFKDr FLJ22582-N 1 MLFICLLCHSDRSRCQDKTVINCIAPLWAFYTSIIEQFNPSLQRLVALGNSYIQAFQALAATSEAYFD * **.*****.* ** *** *. **.**. ..* **

Hs FLJ22582 54 AIQKIGERALQSPTSQILGEILVQMSDTQRHLNSDLEVVVQTFHGGLLQHMEKNTKLDMQFIKDSRQH Mm FLJ22582 54 AIQKIGEQALQSSTSQILGEILVQMSDTQRHLNSDLEVVVQTFHGDLLQHMEKNTKLDMQFIKDSCQH Gg FLJ22582 50 AVEKIGERALQSSTSHVLGEILMQMSDTQRLLSSDLEVVAQTFHVDLLQHMEKNSKMDVQFISESQKQ Dr FLJ22582-N 69 ALSKMGEQAMLSRSSRLLGDVLVQMSDSQRRLTNELEGVFRWFHSEVLQEMDNNVRLDKDYISTSRRR *. *.** *. * .* .**..*.****.** * .**.* ** .* *. * ..* .* *

Hs FLJ22582 122 YELEYRHRAANLEKCMSELWRMERKRDKNVREMKESVNRLHAQMQAFVSESQRAAELEEKRRYRFLAEMm FLJ22582 122 YEIEYRHRAANLEKCMSELWRMERKRDKNAREMKESVNRLHAQMQAFVSESKRAAELEEKRRYRFLAEGg FLJ22582 118 YELEYRRKAKNLEKCTTELWRMERARDKNVREMKENVMRLRSEMQAFLSNSQREAELEEKRRYRFLAEXl FLJ22582-C 1 IPGEQRAAELEEKRRYRFLAEDr FLJ22582-N 137 YEMEVRNQATALER-----------------QMRRGVPQDGSEYVHFLKECQKEALKEEERRYRFLAE **.* * * **.. ....... .... . *. * *. . * ***********

Hs FLJ22582 190 KHLLLSNTFLQFFGRARGMLQNRVLLWKEQSEASRSPSRAHSPGLLGPALGPP-YPSGRLTPTRLDMPMm FLJ22582 190 KHLLLSNTFLQFLGRARGMLQNRVLLWKEQSEASRSPSRAHSPGLLGPALGPP-YPSGRLTPTRLDMP Gg FLJ22582 186 KHQMLYNTLLQFYSRARGMIQTKAPQWKEQLEASRNPSNSHVQGLL-PSSHSQGYPSGRLTPTHLEM- Xl FLJ22582-C 22 KHWLLCSSFVQYHNRAQGLLQARVTPWKEQIDASRS------QTASGP----QNFASSRQASTSRDSV Dr FLJ22582-N 188 KHCGLTQSIAHLMNK ** * . . .* *..* . **** .*** .. . * . * * . * .

Hs FLJ22582 257 PRPLGEFSSPRSRHGSGSYG-TEPDARPASQLEPDRRSLPRTPSASSLYSGSAQSSRSNSFGERP---Mm FLJ22582 257 PRPLGEYGSPRSRHGSGSYGPEPAEARSASQLEPDRRSLPRTPSASSLYASSTQRSRSNSFGERL---Gg FLJ22582 252 -RPLGDFASSLAGSRSSIFSQEPSETRLSPQPEVPRRPLQRTPSASLLPAG--RVSRSGSFGEASSSSXl FLJ22582-C 80 PLPP----RPVDREQN--------DSRASVRSDSSRRGLQRTPSTGSVGS--SQLTRSSSFGEVAVAA . * ... . ..* . . ** * ****. . .** ****

Hs FLJ22582 321 ----GGGGGARRVRALVSHSEGANHTLLRFSAGDVVEVLVPEAQNGWLYGKLEGSSASGWFPEAYVKAMm FLJ22582 322 -----GGGGARRVRALVSHSEGANHTLLRFSAGDVVEVLVPEAQNGWLYGKLEGSSASGWFPEAYVKPGg FLJ22582 317 --EGRRRSGTVRVQAIAPHTTGANRTLLRFDPGDVITVLMPEAQNGWLYGKLEGSSTCGWFPEAYVKPXl FLJ22582-C 134 VAAAAAGAGGRRVQAIVDHNTGGNRTLLQFKKGDLITVLVPEARNGWLYGKLEGLSMNGWFPEAYVKS * ** *. * * * *** * **.. **.*** ********** * *********

Hs FLJ22582 385 LEEGPVNPMTPVTPMTSMTSMSPMTPMTPMNPGNELPSRSYPLRGSHSLDDLLDRPGN----------Mm FLJ22582 385 VEEIPVNPMNPVAPMNSMAPMSPMN---------ELPSRSYPLRGSHSLDDLLDRPGNPTASSEYWDSGg FLJ22582 383 LEDARDME--------------------------EPPVRSFPLRSSHSMDDILDHPSVSSS-------Xl FLJ22582-C 202 LDR-------------------------------DQDSRSFPLRSAHSTGDLLDGSDNFPEPDYRPKP .. . **.*** .** *.**

Hs FLJ22582 443 -----STPSRVPSRAPSPAPPPLP-SSRRSSMGSTAVATDVKKLMSSEQYPPQELFPRGTNPFATVKLMm FLJ22582 444 QSR-SRTPSRVPSRAPSPAPPPLP-SSRRSSVGSMGAATDVKKLMSWEQ-NPPELFPRGTNPFATVKLGg FLJ22582 418 -----SWSAAAQAAVPNPPPPPVGASSHQSGSVTSGG-S--KKPGVFDQ--PPELFPRGTNPFATVKLXl FLJ22582-C 239 QGQGFSTPNSTRGSEANVAPPPPP-SSGNVARKSTGGAR-------------GDLFPGGTNPFATGKL *** ** . . .. . .*** ******* **

Hs FLJ22582 505 RPTITNDRSAPLIR 518Mm FLJ22582 509 RPTVTNDRSAPLIR 522Gg FLJ22582 476 RPTVTNDRSAPIIR 489Xl FLJ22582-C 293 RPTVTNDRSAPLIR 306 ***.*******.**

Supplemental Fig. 3. Alignment of FLJ22582 proteins.

The IMD is boxed. The SH3 domain is shaded. Asterisks show the identity and dots show the similarity. FLJ22582 protins show lower levels of sequence conservation than other members in the family. Species: Hs, human; Mm, mouse; Gg, chicken; Xl, Xenopus laevis; Dr, zebra fish. For detail, see Collection of nucleotide sequence data of putative members of the IRSp53/MIM family proteins.

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Hs MIM 1 MEAVIEKECSALGGLFQTIISDMKGSYPVWEDFINKAGKLQSQLRTTVVAAAAFLDAFQKVADMATNTRGMm MIM 1 MEAVIEKECSALGGLFQTIISDMKGSYPVWEDFINKAGKLQSQLRTTVVAAAAFLDAFQKVADMATNTRGGg MIM 1 MEAVIEKECSALGGLFQTIISDMKGSYPVWEDFINKAGKMQSQLRTTVVAAAAFLDAFQKVADMATNTRGXl MIM 1 MDSSIERECSALGGLFQTIINDMKASYPTWEDFVSKAIKLQSHLKSMIIVTGSFLDAFQKIADMAINTRGHs ABBA-1 1 MET-AEKECGALGGLFQAIVNDMKSSYPIWEDFNSKATKLHSQLRTTVLAAVAFLDAFQKVADMATNTRGMm ABBA-1 1 MET-AEKECGALGGLFQAIVNDMKSSYPIWEDFNSKAAKLHSQLRTTVLAAVAFLDAFQKVADMATNTRGGg ABBA-1 1 -----------LGGLFQAIINDMKSSYPIWEDFNSKATKLHSQLRTTVLAAVAFLDAFQKVADMATNTRGDr ABBA-1 1 MES-VEKECGALGGLFQAIVNDMKYSYPVWEDFITKATKLHSQLRTTVLATAAFLDAFQKVADMATNTRGDr ABBA-2 1 MDAGMEKECSALGGLFQIIMNDMKASYPAWEDFVTKGAKLQSQLRTTIIVTSSFLDAFQKVADIATGTRG *.. *.** ******* *. *** *** **** * *..*.*.. .. . .*******.**.* .***

Hs MIM 71 GTREIGSALTRMCMRHRSIEAKLRQFSSALIDCLINPLQEQMEEWKKVANQLDKDHAKEYKKARQEIKKKMm MIM 71 GTREIGSALTRMCMRHRSIEAKLRQFSSALIDCLINPLQEQMEEWKKVANQLDKDHAKEYKKARQEIKKKGg MIM 71 GTREIGSALTRMCMRHRSIESKLRQFSSALIDCLINPLQEQMEEWKKVANQLDKDHAKEYKKARQEIKKKXl MIM 71 ATKEIGSALTRMCMRHRNIESRLKHLTVALTDSLINPLEGKIEEWKKVANQLDKDHAKEYKKARANIKKKHs ABBA-1 70 ATRDIGSALTRMCMRHRSIETKLRQFTNALLESLINPLQERIEDWKKAANQLDKDHAKEYKRARHEIKKKMm ABBA-1 70 ATRDIGSALTRMCMRHRSIETKLRQFTNALLESLINPLQERIEDWKKSANQLDKDHAKEYKRARHEIKKKGg ABBA-1 60 ATRDIGSALTRMCMRHRSIEAKLRQFTNALMESLINPLQDRIEDWKKTANQLDKDHAKEYKRARHEIKKKDr ABBA-1 70 ATRDIGSALTRMCMRHRSIEAKLRHFTNALMEKMIGPLQDKIEEWKKTAALLDKDHAKEYKRSRQEIKKKDr ABBA-2 71 ATKEIGSALTRMCMRHRSIENKLKLFTTALMDNLITPLELKIEEWKKVASQLDKDHAKEYKKARSEIKKK *..************* ** .*. . ** . .* **. ..*.*** * **********..* ****

Hs MIM 141 SSDTLKLQKKAKK---GRGDIQPQLDSALQDVNDKYLLLEETEKQAVRKALIEERGRFCTFISMLRPVIEMm MIM 141 SSDTLKLQKKAKK---GRGDIQPQLDSALQDVNDKYLLLEETEKQAVRKALIEERGRFCTFISMLRPVIEGg MIM 141 SSDTLKLQKKAKK---GRGDIQPQLDSALQDVNDKYLLLEETEKQAVRKALIEERGRFCTFISMLRPVIEXl MIM 141 SSDTIKLQKKVKK---GKDDARVQLDSALQDVSDKYVLLEETEKKAVYRALVEERGRFCMVVSFPQPVIEHs ABBA-1 140 SSDTLKLQKKARKELLGKGDLQPQLDSALQDVNDMYLLLEETEKQAVRRALIEERGRFCTFITFLQPVVNMm ABBA-1 140 SSDTLKLQKKARKELLGKGDLQPQLDSALQDVNDMYLLLEETEKQAVRRALIEERGRFCTFITFLQPVVNGg ABBA-1 130 SSDTLKLQKKARKELLGKGDLQPQLDNALQDVNDMYLLLEETEKQAVRKALIEERGRFCTFITFLQPVVNDr ABBA-1 140 SSDTIKLQKKARKELPGRGGLQPQLNSAMQDVNDMYLLMEETEKQAVRRALLEERGRYCTFISFLQPVVVDr ABBA-2 141 SSDTIKLQKKVKK---GKDEVRMQLDSAMQDVNDMYLLMEETEKQAVRRALVEERGRFCTFISFLQPVVN ****.***** .* *. . ** .*.*** * *.*.*****.** .**.*****.* .. .**.

Hs MIM 208 EEISMLGEITHLQTISEDLKSLTMDPHKLPSSSEQVILDLKGSDYSWSYQTPPSSPSTTMSRKSSVCSSLMm MIM 208 EEISMLGEITHLQTISEDLKSLTMDPHKLPSSSEQVILDLKGSDYSWSYQTPPSSPSTTMSRKSSVCSSLGg MIM 208 EEISMLGEITHLQTISDDLKSLTMDPHKLPSSSEQVILDLKGSDYSWSYQTPPSSPSTTMSRKSSVCSSLXl MIM 208 EEISMLGEITHLQTISEDLKSLTMDPHKLPSSSEQRILDLKGSDYSWSYQTPPSSPSTTMSRKSSVCSSLHs ABBA-1 210 GELTMLGEITHLQGIIDDLVVLTAEPHKLPPASEQVIKDLKGSDYSWSYQTPPSSPSSSSSRKSSMCSAPMm ABBA-1 210 GELTMLGEITHLQGIIDDLVVLTADPHKLPPASEQVIKDLKGSDYSWSYQTPPSSPSSSNSRKSSMCSAPGg ABBA-1 200 GELTMLGEITHLQGIIDDLVVLTAEPHKLPPASEQVIKDLKGSDYSWSYQTPPSSPSSSSSRKSSMCSSVDr ABBA-1 210 GEIAMLGEATHLQAIIEDLTVLTTDPHKLPEASEQVILDLKGSDYNWSYQTPPSSPSSIGSRKSSLCSL-Dr ABBA-2 208 GEIAMLGEITHLQAIIDDLTVLTSDPHKLPPASEQVIKDLKGSDYSWSYQTPPSSPSSSGSRKSSMCSSV *..**** **** * .** ** .***** *** * ******* ***********. *****.**

Hs MIM 278 NSVNSSDSRSS----GSHSHSPSSHYRYRSSNLAQQAPV-RLSSVSSHDSGFISQDA-FQSKSPSPMPPEAMm MIM 278 NSVNSSDSRSS----GSHSHSPSSHYRYRSSNLAQQAPV-RLSSVSSHDSGFISQDA-FQSKSPSPMPPEAGg MIM 278 NSVNSSDSRSS----GSHSHSPSSHYRYRSSNLPQQAPM-RLSSVSSHDSGFMSQDA-FQSKSPSPMPPEAXl MIM 278 NSVNSSDSRYS----GSHSHSPSSHYRYRSSNLAQKAPV-RLSRGHSH-----------------------Hs ABBA-1 280 SSSSSAKGGGAPWPGGAQTYSPSSTCRYRSLAQPATTTA-RLSSVSSHDSGFVSQDATY-SKPPSPMPSDIMm ABBA-1 280 SSSGSAKGGGAPWPGGAQTYSPSSTCRYRSLAQPATT---RLSSVSSHDSGFVSQDPTY-SKPPSPMPSDIGg ABBA-1 270 SS---AKGGL-PWPGGDr ABBA-1 280 --VH-------LPPGGAH----------------------RLSSVSSHDSGFVSQDANVHSKPPSPMPSDIDr ABBA-2 278 -SAHSSASRSS---GGSQTHSPSSSCRYRSLAQPLTTAAHRLSSVSSHDSGFISQDANVYSKPPSPMPSDI *....** *.** **... ****.***** *** . *************.***** *********.

Hs MIM 343 PNQLSNGFSHYSLSSESHVGPTGAGLFPHCLPASRLLPRVTSVHLPDYAHYYTIGPGMF----PSSQIPSMm MIM 343 ANQLSNGFSHCSLSSESHAGPVGAGPFPHCLPASRLLPRVTSVHLPDYAHYYTIGPGMF----PSSQIPSGg MIM 343 PNQLSNGFYHCSLSSDPSVASVGAGPFPHFPPVSRAWTRAPSALLPDYVHYYTIGPGML----PSSKIPSHs ABBA-1 349 TSQKSSSSASSEASETCQSVS--------------------------------------ECSSPTS----Mm ABBA-1 347 TSQKSSSSASSEASETCQSVS--------------------------------------ECSSPTS----Dr ABBA-1 3194TSQKSSSSASSEASETCQSVS--------------------------------------ECGSPTIQ---Dr ABBA-2 346 TSQKSSSSASSEASETCQSVS--------------------------------------ECSSPTT---- **.*.** * *****... ****.***** *** . *************.*****. *****. **

Supplemental Fig. 4. Alignment of MIM sub-family proteins.

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Supplemental Fig. 4. Alignment of MIM sub-family proteins (page 2).

The IMD is boxed. The WH2 domain is underlined. Asterisks show the identity and dots show the similarity. MIM and ABBA are highly homologous, but characteristic amino acid differences are noted even in highly conserved regions (shaded residues in MIM sequences). The N-terminus of chicken ABBA-1 could not be retrieved. Two zebra fish cDNAs show the ABBA characters. Species: Hs, human; Mm, mouse; Gg, chicken; Xl, Xenopus laevis; Dr, zebrafish. For detail, see Collection of nucleotide sequence data of putative members of the IRSp53/MIM family proteins.

Hs MIM 473 MEACEELALALSRGLQLDT-QRSSRDSLQCSSGYSTQTTTPCCSEDTIPSQVS----------DYDYFSVMm MIM 473 MAACEELTLALSRGLQLDV-QRSSRDSLQCSSGYSTQTTTPCCSEDTIPSQVS----------DYDYFSVGg MIM 470 MEPCEELALALTRGLQLDT-QRSSRDSLQCSSGYSTQTTTPCCSEDTIPSQVS----------DYDYFSVXl MIM-C 621 RLDRSGIPCI-------Hs ABBA-1 441 SPAASDLAMVLTRGLSLEH-QKSSRDSLQYSSGYSTQTTTPSCSEDTIPSQGS----------DYDCYSVMm ABBA-1 439 SPAASDLAMVLTRGLSLEH-QKSSRDSLQYSSGYSTQTTTPSCSEDTIPSQGS----------DYDCYSVDr ABBA-1 4061SSAASELAMVLTRGLSIEQTQKSSCDSLQYSSGYSTQNTTPSCSEDTIPSQGS----------EYDCYSMDr ABBA-2 429 SPAASDLAMVLTRGLSMEQ-QKSSRDSLQYSSGYSTQTTTPSCSEDTIPSQGS----------DYDGYSV ...**.*..**.***. . **.*******.*********************.* .**..*.

Hs MIM 532 SGDQEAD-QQEFDKSSTIPRNSDISQSYRRMFQAKRPASTAGLPTTLG----------PAMVT-PGVATIMm MIM 532 SGDQEAE-QQEFDKSSTIPRNSDISQSYRRMFQAKRPASTAGLPTTLG----------PAMVT-PGVATIGg MIM 529 SGDQEAE-QQEFDKSSTIPRNSDISQSYRRMFQTKRPASTAGLPTTLG----------PVIVT-PGVATIXl MIM-C 611 ---QEGE-QQDFDKSSTIPRNSDISQSYRRMFQAKRPASTAGLPANFG----------PVIVT-PGVATIHs ABBA-1 500 NGDADSEGPPEFDKSSTIPRNSNIAQNYRRLIQTKRPASTAGLPTA-GL---PTATGLPS-GAPPGVATIMm ABBA-1 498 NGDADSEGPPEFDKSSTIPRNSNIAQNYRRLIQTKRPASTAGLPTA-GL---PTAMGLPS-GAPPGVATIDr ABBA-1 466 NGDADNDTQTDFDKSSTVPRHSNLAQNYRRMIQTKRPASTAGL---HGALAGPTTNGKGSSIIASGTATIDr ABBA-2 488 NGDTEADGQAEFDKSSTIPRHSNIAQNYRRMIQTKRPASTAGLPSGVGALGGPPGGGGGI----PGTATI .** . . .******.**.* ..*.***. * **********. * * ***

Hs MIM 590 RRTPSTKPSVRRGTIGAGPIPIKTPVIPVKTPTVPDLPGVLPAP--PDGPEERGEHSPESPS--VGEGPQMm MIM 590 RRTPSTKPSVRRGTIGAGPIPIKTPVIPVKTPTVPDLPGVLPSP--PDGPEERGEHSPESPS--AGEGPQGg MIM 587 RRTPSTKPSVRRGTIGAGPIPIKTPVIPVKTPTVPDIPGGLPGA--LAGTEECPEQSPESPA--AGDGGQXl MIM-C 66 RRTPSTKPSVRRGTIGGGPIPIKTPVIPVKTPTVPDLPGGLPSS----PSEDYGEQSPDSPR--ATESSTHs ABBA-1 565 RRTPSTKPTVRRALSSAGPIPIRPPIVPVKTPTVPDSPGYMGPT--RAGSEECVFYTDETASPLAPDLAKMm ABBA-1 563 RRTPSTKPTVRRALSSAGPIPIRPPIVPVKTPTVPDSPGYVGPT--RAGSEECVFYTDEVASPLAPDLAKDr ABBA-1 533 RRTPSSKTNVRRTPSTVGPIPIRPPIVPVKTPTVPDSPGFPSPPPEHNGSEESLYN-DDSME--ILEY-KDr ABBA-2 554 RRTPSTKPGVRRTLSNAGPIPIRPPIVPVKTPTVPDSPGGSTGPPARVGSEECVFYVPDDASPGALEYVK *****.* *** *****. *..********* ** *. . .

Hs MIM 656 GVTS---MPSSMWSGQASVNPPLPGPKPSIP--EEHRQAIPESEAEDQEREP----------------PSMm MIM 656 GVSN---IPSSLWSGQAPVNPPLPGPKPSIP--EEHRQAIPESEAEDQERDP----------------PSGg MIM 653 SVPS---VPSSSWSGQASVNPPPSSQKLGAA--DEQRPAVPESEGEESERDG-----------------AXl MIM-C 130 LTSD---MPSSLWSGQASVNPPVPATQGLVL--EEQRQTRSENDEEEGE---------------QVNSPTHs ABBA-1 633 ASPKRLSLPNTAWGSPSPEAAGYPGAGA-----EDEQQQLAANRHSLVEKLGELVAGAHALGEGQFPFPTMm ABBA-1 631 ASPKRLSLPNTAWGSQSPEVASYGGGAAVGLATEDEEQQLAANRHSLVEKLGELVAGAHALGEGQFPFPTDr ABBA-1 599 ASPKRMSLPT--WGSGGGSVYSQPPGAAGF-SSEEDQ-ILAANRHSLVEKIGELVANAHALGEGQFPFPADr ABBA-2 624 ASPKRLSLPNTAWGSGGVGGLEMSVYGQPG---EEDH-LLAANRHSLVEKIGELVASAHALGEGQFPFPS .* . * .. *. *

Hs MIM 705 ATVSPGQ--IPESDPADLS-----PRDTPQGEDMLNAIRRGVKLKKTTTNDRSAPRFS 755Mm MIM 705 ATVSPGP--IPESDPADLS-----PRESPQGEDMLNAIRRGVKLKKTTTNDRSAPRFS 755Gg MIM 701 SSLAPAGQ--PELDPGDLS-----PGDVPQGEDMLNAIRRGVKLKKTTTNDRSAPRISN 751Xl MIM-C 180 A--DPTAQ--AEPEPGEA----------VQGENMLHSIRRGVKLKKTLTNDRSAPRLS 223Hs ABBA-1 698 ALSATPTEETPTPPPAATS-------DPPA-EDMLVAIRRGVRLRRTVTNDRSAPRIL 747Mm ABBA-1 701 ALSATPSEETPTPPPAATS-------DPPA-EDMLVAIRRGVRLRRTVTNDRSAPRIL 750Dr ABBA-1 6653-------------DPLADQSLQQDEGHEKGGEDMLKTIRRGVRLRKVSSNDRSAPRIMR 710Dr ABBA-2 6909L--------PNQPPPG-PEAQVDPEAAQEEG-DMLKSIRRGVRLRKTVSNDRSAPRIL 737 * .* ** .*****.*.. .*******

Hs MIM 409 WKDWAKPGPYDQPLVNTLQRRKEKREP-DPNGGGPTTASGPPAA--AEEAQRP-RSMTV-SAATRPGEE-Mm MIM 409 WKDWAKPGPYDQPLVNTLQRRKEKREP-DSNGGGPTTTGGPPAG--AEEAQRP-RSMTV-SAATRPGEE-Gg MIM 409 WKDWAKPGPYDQPMVNTLQRRKEKREA-DLNGG---AQSGPPVP---EEAQRP-RSMTV-SAATRQGEE-Hs ABBA-1 377 --DWSKVGSHEQPSGATLQRRKDRVELLRDTEPGPASGGTLGPS--GEEAPRP-R-MSPATIAAKHGEEVMm ABBA-1 375 --DWTKAGPHEQPSATTLQRRKDRVEHLRDTEPGPTGGGTVGSS--GEEVPRT-R-MSPATIAAKHGEEVDr ABBA-1 348 --DWSKVGQYEQLSVGQ-QRRTEAAE--------PSSGAGGHTHTHPEDTYRLAR-MNSKDATAKHGKLVDr ABBA-2 374 --DWSKVGSSDQPLASTLQRRKEPV-----AETSP---QSQGYS---EDPQRP-R-MTPATIAAKHGEEV ** *****.***...*****.*****. *** * ************..*****.***** * .*.

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Supplemental Fig. 5. Amino acid seequence alignment of the IRSp53/MIM homology domains.

S

Hs IRSp53 1 MSLSR-SEEMHRLTENVYKT--IME--QFNPSLRNFIAMGKNYEKALAGVTYAAKGYFDALVKMGELASESQG-SKELGDVLFQMAEGg IRSp53 1 MAR-SEEVHRLTENVYKT--IME--QFNPSLRNFISMGKTYEKALASMTYAAKGYFDALVKMGELASESQG-SKELGDVLFQMAEDr IRSp53 1 MSR TDEVHRITENVYKS--IME--QFNPCLRNFVAMGKTYEKALSSVTFAAKGYFDALVRMGELASESHG-SKDLEDVLFQMAEHs IRTKS 1 ME--QFNPGLRNLINLGKNYEKAVNAMILAGKAYYDGVAKIGEIATGSPV-STELGHVLIEISSGg IRTKS 1 ME--QFNPGLRNLINLGKNYEKAVNAMVVAGRSYYDSLAKIGDISADSPV-SKELGQVLVEISRDr IRTKS 1 MSRASDEVNKLTESTYKN--VME--QFNPGLRNLVNLGKNYEKSVAAMTFAGKAYFDALSKIGESAAVSPV-SRELGVVLMEISEHs FLJ22582 1 -

Dr FLJ22582 1 MLFICLLCHSDRSRCQDKTVINCIAPLWAFYT-IIE—-QFNPALENLVYLGNNYLRAFHALSEAAEVYFSAIQKIGERALQSP-TSRLLGDVLVQMSDDmCG32082 1 MNISIDRFFFELQNILD--RFNPGARQLIAAGKSYLKALHGAATASRLFNEALAKIAMNAQQS-G-TGDIGSALMSVVN

Hs MIM 1 MEAVIEKECSALGGLFQTIISDMKGSYPVWEDFINKAGKLQSQLRTTVVAAAAFLDAFQKVADMATNTRGGTREIGSALTRMCMGg MIM 1 -MEAVIEKECSALGGLFQTIISDMKGSYPVWEDFINKAGKMQSQLRTTVVAAAAFLDAFQKVADMATNTRGGTREIGSALTRMCMHs ABBA-1 1 -META-EKECGALGGLFQAIVNDMKSSYPIWEDFNSKATKLHSQLRTTVLAAVAFLDAFQKVADMATNTRGATRDIGSALTRMCM

Dr ABBA-1 1 -MES-VEKECGALGGLFQAIVNDMKYSYPVWEDFITKATKLHSQLRTTVLATAAFLDAFQKVADMATNTRGATRDIGSALTRMCMDr ABBA-2 1 -MDAGMEKECSALGGLFQIIMNDMKASYPAWEDFVTKGAKLQSQLRTTIIVTSSFLDAFQKVADIATGTRGATKEIGSALTRMCMCe M04F3.5 1 -MDP--DAEFNLLTTLYQSVVHDMKHTYPGWDALSQKAQKLGTQLRATAQCLSTFTEAMQGVCDNANNLKGASRDVGACVTRVCI

Hs IRSp53 82 VHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTE-QRSKGDALDKCQAEL-KKLRKKSQ-----GSKNPQKYSDKELQYIDAISNKGg IRSp53 80 VHRQIQNQLEEMLKSFHNELLTQLEQKVELDSRYLSAALKKYQTE-QRNKGDSLDKCQAEL-KKLRKKSQ-----GSKNPQKYSDKELQYIEAISNKDr IRSp53 80 VHRQIQMQLEDMLKSFHNELLTELEKKVELDVRYLNAALKKYQME-HKSKGESLEKCQAEL-KKLRRKSQ-----GSKNPSKYGDKEMQYVETISSKHs IRTKS 81 THKKLNESLDENFKKFHKEIIHELEKKIELDVKYMNATLKRYQTE-HKNKLESLEKSQAEL-KKIRRKSQ-----GSRNALKYEHKEIEYVETVTSRGg IRTKS 62 THKKLNDSLEESFKKFHKEIISELEKKIDLDVKYMTATLKRYQTE-HRSKLDSLEKSQAEL-KKIRRKSQ-----GARNVMKYEHKEMEYLETVSSRDr IRTKS 81 VHKNAQLEMEESFKRFHKELIAELEKKTDMDTKYMTATFKRYQSE-HKLKQDFLDKSQADL-KKLRRKSQ-----G-KHSSKYEIKENECMETISSRHs FLJ22582 81

Dr FLJ22582 96 SQRRLTNELEGVFRWFHSELLQEMDNNVRLDKDYISTSRRRYEMEVRNQ-ATALER--------------------------------QMRRGVPQDDmCG32082 76 VNKEIQDQQMNILKAFYVDVLVPLETNLEKDTKVVQHEQKKFLQQ-HKVRMESYQKAVSTM-KKQRKKKA--------TPEN-TEKELRSLQLLEDQ

Hs MIM 85 RHRSIEAKLRQFSSALIDCLINPLQEQMEEWKKVANQLDKDHAKEYKKARQEIKKKSSDTL-KLQKKAKK---GRGDIQPQL-DSALQDVNDKYLLLGg MIM 85 RHRSIESKLRQFSSALIDCLINPLQEQMEEWKKVANQLDKDHAKEYKKARQEIKKKSSDTL-KLQKKAKK---GRGDIQPQL-DSALQDVNDKYLLLHs ABBA-1 84 RHRSIETKLRQFTNALLESLINPLQERIEDWKKAANQLDKDHAKEYKRARHEIKKKSSDTL-KLQKKARKELLGKGDLQPQL-DSALQDVNDMYLLL

Hs IRSp53 172 QGELENYVSDGYKTALTEERRRFC-FLVEKQCAVAKNSAAYHSKGKELLAQKLPLWQQACA--------------DPSKIPERAVQLMQQVAS 248Gg IRSp53 170 QGELENYVSDGYKTALTEERRRFC-FLVEKQCAVAKSAITYHAKGKEMLTQKLPLWQQSCS--------------DPNKIPDRAIQLMQQMAA 248Dr IRSp53 170 QNELDNYISESYKNALSEERRRYC-FLVDRQCAVAKTSNTYHTKGRDLLTQKIPLWQQACA--------------DPNKLPERAMLLAQQMSS 248Hs IRTKS 171 QSEIQKFIADGCKEALLEEKRRFC-FLVDKHCGFANHIHYYHLQSAELLNSKLPRWQETCV--------------DAIKVPEKIMNMIEEIKT- 248Gg IRTKS 152 QTDIQRFIAEGCREALLEEKRRFC-FLVDKHCSFTQHMHFYHAQCAEFLNAKLPGWQETCS--------------DATKVPEKVKMMIEEIRT- 229Dr IRTKS 170 QTDMQRFIADGCKEALLEEKRRFC-FLVDKHCALSYQLSAFHDKAKEMLTLKLPSWQEKCN--------------DATRVPDTVISMIEGLRT- 247Hs FLJ22582 162 HAQMQAFVSESQRAAELEEKRRYR-FLAEKHLLLSNTFLQFFGRARGMLQNRVLLWKEQS---------------EASRSPSRAHSPGLLGPAL 239

Dr FLJ22582 160 GSEYVHFLKECQKEALKEEERRYR-FLAEKHCGLTQSIAHLMNK 202DmCG32082 162 KKKLDVFCDQSYKNAMTQERRRYG-FVLERQCSIAKHWMVYHTTGKTVIDNNFENWQEIAA------------SREI--IPPAAYESGYSTSN 239

Hs MIM 177 EETEKQ----AVRKALIEERGRFCTFISMLRPVIEEEISMLGEITH--LQTISEDLKSLTM--------------DPHKLPSSSEQVILDLKGS-249Gg MIM 177 EETEKQ----AVRKALIEERGRFCTFISMLRPVIEEEISMLGEITH--LQTISDDLKSLTM--------------DPHKLPSSSEQVILDLKGS-249Hs ABBA-1 179 EETEKQ----AVRRALIEERGRFCTFITFLQPVVNGELTMLGEITH--LQGIIDDLVVLTA--------------EPHKLPPASEQVIKDLKGS-251

Dr ABBA-1 176 EETEKQ----AVRRALLEERGRYCTFISFLQPVVVGEIAMLGEATH--LQAIIEDLTVLTT--------------DPHKLPEASEQVILDLKGS--241Dr ABBA-2 177 EETEKQ----AVRRALVEERGRFCTFISFLQPVLNGEIAMLGEITH--LQAIIDDLTVLTS--------------EPHKLPPASEQVIKDLKGS-249Ce M04F3.5 148 ---------EICTKVLTEQRTQFSFFIGTLMPVLNAQMNMLDEGAH--LRQVVDNLDS-TVKHVDTEQLVSSIVSDIAQGPDSAWKQCLAQAGN—228

Dr ABBA-1 84 RHRSIEAKLRHFTNALMEKMIGPLQDKIEEWKKTAALLDKDHAKEYKRSRQEIKKKSSDTI-KLQKKARKELPGRGGLQPQL-NSAMQDVNDMYLLMDr ABBA-2 85 RHRSIENKLKLFTTALMDNLITPLELKIEEWKKVASQLDKDHAKEYKKARSEIKKKSSDTI-KLQKKVKK---GKDEVRMQL-DSAMQDVNDMYLLMCe M04F3.5 83 KQRAIENRMRSWADALCEELAISLQQRGNYWKSRCHDMDKTAAKHVKKVR--------------SRKQR----------PDV--TAMNEQR------

G

N

GN

MSRGPEEVNRLTESTYRN-V

Helix I Helix II Helix III

Helix IV

Helix V

RSEMQAFLSNSQREAELEEKRRYR-FLAEKHQMLYNTLLQFYSRARGMIQTKAPQWKEQL---------------EASRNPSNSHVQGLL-PSS

R YKS--IME—-QFNPALENLVYLGNNYLRAFHALSEAAEVYFSAIQKIGERALQSP-TSQILGEILVQMSD

TQRHLNSDLEVVVQTFHGGLLQHMEKNTKLDMQFIKDSRQHYELEYRH-RAANLEKCMSELWRMERKR---------------DKNVREMKESVNRL

MDLSYRSTISIYKS-ILE--QFNPALENLVYLGNNYLRAFHALSKAAEVYFKAVEKIGERALQSS-TSHVLGEILMQMSD

TQRLLSSDLEVVAQTFHVDLLQHMEKNSKMDVQFISESQKQYELEYRR-KAKNLEKCTTELWRMERAR---------------DKNVREMKENVMRL

Gg FLJ22582 1 -MAPEMDQFY STMAI

Gg FLJ22582 77

Gg FLJ22582 158

-

Species used: Hs, human; Gg, chicken; Dr, zebra fishi; Dm, D. melanogaster; Ce, C. elegans. Red lines are the consensus helicespredicted and black dots and lines represent conserved amino acid residues. Residues color codes: acidic, red; basic, blue; alcoholic, light blue; aromatic, brown; polar, green; P, black; C, yellow; non-polar, plain. The insertion of four amino acids in Dr IRSp53 (see Supplemental Fig. 1) is not shown.

234

Gg ABBA-1 1 LGGLFQAIINDMKSSYPIWEDFNSKAGKLHSQLRTTVLAAVAFLDAFQKVADMATNTRGATRDIGSALTRMCM

Gg ABBA-1 74 RHRSIEAKLRQFTNALMESLINPLQDRIEDWKKTANQLDKDHAKEYKRARHEIKKKSSDTL-KLQKKARKELLGKGDLQPQL-DNALQDVNDMYLLL

Gg ABBA-1 169 EETEKQ----AVRKALIEERGRFCTFITFLQPVVNGELTMLGEITH--LQGIIDDLVVLTA--------------EPHKLPPASEQVIKDLKGS-241

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Hs GgDrHsGgXlHsGgXlHsDrDrWH2 consensus

GENPFAA----VKLRPTVTNDRSAPIIR GENPFAA----VKLRPTVTNDRSAPIIRN GGNPFAT----VRLRPTVTNDRSAPII GTNPFAT----VKLRPTITNDRSAPLIR GTNPFAT----VKLRPTVTNDRSAPIIR GTNPFAT----GKLRPTVTNDRSAPLIRQGEDMLNAIRRGVKLKKTTTNDRSAPRFSQGEDMLNAIRRGVKLKKTTTNDRSAPRISNQGENMLHSIRRGVKLKKTLTNDRSAPRLSPAEDMLVAIRRGVRLRRTVTNDRSAPRILGGEDMLKTIRRGVRLRKVSSNDRSAPRIMREG-DMLKSIRRGVRLRKTVSNDRSAPRILARSALLAAIRQGKKLKKV

IRTKS IRTKSIRTKSFLJ22582FLJ22582FLJ22582MIMMIMMIMABBA-1ABBA-1ABBA-2

Supplemental Fig. 6. Amino acid sequence homology in the C-terminus of the IRSp53/MIM family proteins.

Species: Hs, human; Gg, chicken; Xl, Xenopus laevis; Dr, Zebra fish

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IRSp53

IRTKS

FLJ22582

MIM

ABBA-1

LocusID

10458

55971

80115

9788

92154

Chromosomalposition

17q25

7q22.1

22q13.1

8p22

16q22.1

pI

9.0

8.8

9.2

8.5

8.8

Gene symbol

BAIAP2

LOC55971

FLJ22582

MTSS1

LOC92154

84%

82%

84%

99%

99%

Helical contentsin IMD

Homologybetween proteins

40/20%

24/20%

55/11%

-

b

b

c

- -

46/28%

28/29%

22/18%

17/21%

b

b

b

b

22/18%

17/21%

14/19%

-

77/11%

c

c

c

c

Homology between IMDs

aC-terminal half; bComparison to IRSp53, identity/similarity; cComparison to MIM.

The IMD containing proteins in the human genome.Supplemental Table 1.

Model protein

NP_059344

NP_061330

NP_079321

NP_055566

XP_208937a

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