Two-dimensional electrophoretic analysis of mixed lineage kinase 2 N-terminal domain binding proteins.
The mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein-protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N-terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S-transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S-radiolabeled MDA-MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2-DE, proteins of Mr 55,000, 31,500 and 34,000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2-DE and subjected to in-gel digestion and capillary or microbore reverse-phase high performance liquid chromatography (RP-HPLC). Resultant peptides were analyzed by peptide mass fingerprinting, N-terminal Edman degradation or tandem mass spectrometry. The 55,000 protein was identified as the cytoskeletal protein, beta-tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti-tubulin antibodies. The 31,500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.
Rasmussen
R K
RK
Trescowthick Research Center, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Ji
H
H
Eddes
J S
JS
Moritz
R L
RL
Reid
G E
GE
Simpson
R J
RJ
Dorow
D S
DS
D000595:Amino Acid Sequence; D001943:Breast Neoplasms; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006801:Humans; D020930:MAP Kinase Kinase Kinases; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis*; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
D009363:Neoplasm Proteins; D011506:Proteins; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C093290:MAP3K10 protein, human