Two-dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2

被引:42
作者
Rasmussen, RK
Ji, H
Eddes, JS
Moritz, RL
Reid, GE
Simpson, RJ
Dorow, DS
机构
[1] PETER MACCALLUM CANC INST,TRESCOWTHICK RES CTR,MELBOURNE,VIC 3000,AUSTRALIA
[2] LUDWIG INST CANC RES,JOINT PROTEIN STRUCT LAB,PARKVILLE,VIC,AUSTRALIA
[3] ROYAL MELBOURNE HOSP,WALTER & ELIZA HALL INST MED RES,PARKVILLE,VIC 3050,AUSTRALIA
关键词
two-dimensional gel protein database; human breast carcinoma proteins; protein kinases; signal transduction components; SH3 binding proteins; mixed lineage kinases; tandem mass spectrometry; protein identification;
D O I
10.1002/elps.1150180342
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et nl. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif: Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction, To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), Including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employ ed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of S-35-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (M-r) of similar to 31 500 and similar to 34 000, bound consistently to the MLK2N protein. To establish accurately the M-r/isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (similar to 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI) - mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins has been constructed and can be accessed via the World Wide Web (URL address: http://www.ludwig.edu.au/www/jpsl/jpslhome.html).
引用
收藏
页码:588 / 598
页数:11
相关论文
共 31 条
[1]   CHARACTERIZATION BY TANDEM MASS-SPECTROMETRY OF STRUCTURAL MODIFICATIONS IN PROTEINS [J].
BIEMANN, K ;
SCOBLE, HA .
SCIENCE, 1987, 237 (4818) :992-998
[2]   CONSTRUCTION OF VALIDATED, NONREDUNDANT COMPOSITE PROTEIN-SEQUENCE DATABASES [J].
BLEASBY, AJ ;
WOOTTON, JC .
PROTEIN ENGINEERING, 1990, 3 (03) :153-159
[3]   A CONSERVED BINDING MOTIF DEFINES NUMEROUS CANDIDATE TARGET PROTEINS FOR BOTH CDC42 AND RAC GTPASES [J].
BURBELO, PD ;
DRECHSEL, D ;
HALL, A .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (49) :29071-29074
[4]   BREAST TUMOR-CELL LINES FROM PLEURAL EFFUSIONS [J].
CAILLEAU, R ;
YOUNG, R ;
OLIVE, M ;
REEVES, WJ .
JOURNAL OF THE NATIONAL CANCER INSTITUTE, 1974, 53 (03) :661-674
[5]   The human keratinocyte two-dimensional gel protein database (update 1995): Mapping components of signal transduction pathways [J].
Celis, JE ;
Rasmussen, HH ;
Gromov, P ;
Olsen, E ;
Madsen, P ;
Leffers, H ;
Honore, B ;
Dejgaard, K ;
Vorum, H ;
Kristensen, DB ;
Ostergaard, M ;
Haunso, A ;
Jensen, NA ;
Celis, A ;
Basse, B ;
Lauridsen, JB ;
Ratz, GP ;
Andersen, AH ;
Walbum, E ;
Kjaergaard, I ;
Andersen, I ;
Puype, M ;
VanDamme, J ;
Vandekerckhove, J .
ELECTROPHORESIS, 1995, 16 (12) :2177-+
[6]   COMPLETE NUCLEOTIDE-SEQUENCE, EXPRESSION, AND CHROMOSOMAL LOCALIZATION OF HUMAN MIXED-LINEAGE KINASE-2 [J].
DOROW, DS ;
DEVEREUX, L ;
TU, GF ;
PRICE, G ;
NICHOLL, JK ;
SUTHERLAND, GR ;
SIMPSON, RJ .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1995, 234 (02) :492-500
[7]   IDENTIFICATION OF A NEW FAMILY OF HUMAN EPITHELIAL PROTEIN-KINASES CONTAINING 2 LEUCINE ISOLEUCINE-ZIPPER DOMAINS [J].
DOROW, DS ;
DEVEREUX, L ;
DIETZSCH, E ;
DEKRETSER, T .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1993, 213 (02) :701-710
[8]   AN APPROACH TO CORRELATE TANDEM MASS-SPECTRAL DATA OF PEPTIDES WITH AMINO-ACID-SEQUENCES IN A PROTEIN DATABASE [J].
ENG, JK ;
MCCORMACK, AL ;
YATES, JR .
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, 1994, 5 (11) :976-989
[9]   SOLUBILIZATION AND PURIFICATION OF ENZYMATICALLY ACTIVE GLUTATHIONE-S-TRANSFERASE (PGEX) FUSION PROTEINS [J].
FRANGIONI, JV ;
NEEL, BG .
ANALYTICAL BIOCHEMISTRY, 1993, 210 (01) :179-187
[10]   ANALYSIS OF PROTEINS FROM HUMAN BREAST EPITHELIAL-CELLS USING 2-DIMENSIONAL GEL-ELECTROPHORESIS [J].
GIOMETTI, CS ;
TOLLAKSEN, SL ;
CHUBB, C ;
WILLIAMS, C ;
HUBERMAN, E .
ELECTROPHORESIS, 1995, 16 (07) :1215-1224