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Two‐dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2
Author(s) -
Rasmussen Richele K.,
Ji Hong,
Eddes James S.,
Moritz Robert L.,
Reid Gavin E.,
Simpson Richard J.,
Dorow Donna S.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180342
Subject(s) - sh3 domain , leucine zipper , electroblotting , edman degradation , biochemistry , biology , fusion protein , microbiology and biotechnology , peptide sequence , kinase , proto oncogene tyrosine protein kinase src , chemistry , gene , recombinant dna , blot
Abstract MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (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 employed in an affinity approach to isolate MLK2‐SH3 domain binding proteins from lysates of 35 S‐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 ˜ 31 500 and ˜ 34 000, bound consistently to the MLK2N protein. To establish accurately the M r / isoelectric point (p I ) 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 (˜ 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: www.ludwig.edu.au/www/jpsl/jpslhome.html ).