title: Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.
abstract: We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.
authors.affiliation: Division of Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Tuoen
authors.initials: T
authors.lastname: Liu
authors.forename: Allison
authors.initials: A
authors.lastname: Dean
authors.forename: Saint
authors.initials: S
authors.lastname: Ashwini
authors.forename: Peter P
authors.initials: PP
authors.lastname: Sheridan
authors.forename: Alok
authors.initials: A
authors.lastname: Bhushan
authors.forename: James C K
authors.initials: JC
authors.lastname: Lai
authors.forename: Shousong
authors.initials: S
authors.lastname: Cao
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Daniels
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D002945:Cisplatin / Q000633:toxicity; D016208:Databases, Factual; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007939:Leukemia L1210 / Q000378:metabolism / Q000473:pathology; D008727:Methotrexate / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D011994:Recombinant Proteins; C063842:dnaK protein, E coli; D002945:Cisplatin; D008727:Methotrexate