title: Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.
abstract: We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.
authors.affiliation: Department of Internal Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, United States.
authors.forename: Tuoen
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID, United States.
authors.forename: Ratan
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID, United States.
authors.forename: Zechary
authors.initials: Z
authors.lastname: Rios
authors.affiliation: Department of Pharmaceutical Sciences, Jefferson School of Pharmacy, Thomas Jefferson University, Philadelphia, PA, United States.
authors.forename: Alok
authors.initials: A
authors.lastname: Bhushan
authors.affiliation: Gynecological Department, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China.
authors.forename: Mengxiong
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, Idaho State University, Pocatello, ID, United States; The ISU Biomedical Research Institute, Idaho State University, Pocatello, ID, United States.
authors.forename: Peter P
authors.initials: PP
authors.lastname: Sheridan
authors.affiliation: Department of Biological Sciences, Idaho State University, Pocatello, ID, United States; The ISU Biomedical Research Institute, Idaho State University, Pocatello, ID, United States.
authors.forename: Shawn E
authors.initials: SE
authors.lastname: Bearden
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID, United States; The ISU Biomedical Research Institute, Idaho State University, Pocatello, ID, United States.
authors.forename: James C K
authors.initials: JCK
authors.lastname: Lai
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID, United States.
authors.forename: Senyo
authors.initials: S
authors.lastname: Agbenowu
authors.affiliation: Chifeng Saliont Pharmaceutical Co., Ltd., 228 North Qinghe Road, Hongshan District, Chifeng, Inner Mongolia Autonomous Region 024000, China. Electronic address: shousongc@gmail.com.
authors.forename: Shousong
authors.initials: S
authors.lastname: Cao
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University, Pocatello, ID, United States; The ISU Biomedical Research Institute, Idaho State University, Pocatello, ID, United States. Electronic address: cdaniels@pharmacy.isu.edu.
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Daniels
mesh_terms: D000818:Animals; D019008:Drug Resistance, Neoplasm; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D007939:Leukemia L1210 / Q000188:drug therapy* / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D010766:Phosphorylation; D058978:Reduced Folate Carrier Protein / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D050883:HSC70 Heat-Shock Proteins; D058978:Reduced Folate Carrier Protein; D014443:Tyrosine; D008727:Methotrexate
keywords: Cross resistance; Heat shock cognate protein 70; Methotrexate binding protein; Methotrexate transportation; Reduced folate carrier; Tyrosine phosphorylation