2-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF HUMAN BREAST-CARCINOMA PROTEINS - MAPPING OF PROTEINS THAT BIND TO THE SH3 DOMAIN OF MIXED LINEAGE KINASE MLK2

MLK2, a member of the mixed lineage kinase (MLK) family of protein kin ases, first reported by Dorow et nl. (Eur. J. Biochem. 1993, 213, 701- 710), comprises several distinct structural domains including an src h omology-3 (SH3) domain, a kinase catalytic domain, a unique domain con taining two leucine zipper motifs, a polybasic sequence, and a cdc42/r ac interactive binding motif: Each of these domains has been shown in other systems to be associated with a specific type of protein interac tion in the regulation of cellular signal transduction, To study the r ole of MLK2 in recruiting specific substrates, we constructed a recomb inant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), In cluding the SH3 domain (residues 23-77), fused to glutathione S-transf erase. 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. Electro phoretic analysis of bound proteins revealed that two low-abundance pr oteins with a molecular weights (M-r) of similar to 31 500 and similar to 34 000, bound consistently to the MLK2N protein. To establish accu rately the M-r/isoelectric point (pI) loci of these MLK2-SH3 domain bi nding proteins, a number of abundant proteins in a two-dimensional ele ctrophoresis (2-DE) master gel were identified to serve as triangulati on marker points. Proteins were identified by (i) direct Edman degrada tion following electroblotting onto polyvinylidene difluoride (PVDF) m embranes, (ii) Edman degradation of peptides generated by in-gel prote olysis and fractionation by rapid (similar to 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HP LC), (iii) mass spectrometric methods including peptide-mass fingerpri nting and electrospray (ESI) - mass spectrometry (MS)-MS utilizing cap illary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analy sis. Using this information, a preliminary 2-DE protein database for t he human breast carcinoma cell line MDA-MB231, comprising 21 identifie d proteins has been constructed and can be accessed via the World Wide Web (URL address: http://www.ludwig.edu.au/www/jpsl/jpslhome.html).