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

Rasmussen, RK; Ji, H; Eddes, JS; Moritz, RL; Reid, GE; Simpson, RJ; Dorow, DS

Author(s): Rasmussen, RK; Ji, H; Eddes, JS; Moritz, RL; Reid, GE; Simpson, RJ; Dorow, DS;
Details: Publication Year 1997-03,Volume 18,Issue #3-4,Page 588-598
Journal Title: ELECTROPHORESIS
Publication Type: Journal Article
Abstract: 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).
Publisher: VCH PUBLISHERS INC
Keywords: AMINO-ACID-SEQUENCES; GEL-ELECTROPHORESIS; MASS-SPECTROMETRY; PEPTIDES; DATABASE; IDENTIFICATION; EXPRESSION; SPECTRA; FAMILY
Publisher's Version: https://doi.org/10.1002/elps.1150180342
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 1997-03-01 12:00:00