title: Calpain inhibitor causes accumulation of ubiquitinated P-glycoprotein at the cell surface: possible role of calpain in P-glycoprotein turnover.
abstract: P-glycoprotein (Pgp) is a plasma-membrane glycoprotein that confers multi-drug resistance (MDR) on cells and displays ATP-driven drug pumping. The possible contribution of calpain-mediated proteolytic pathways to the functional regulation of the Pgp molecule was evaluated using K562/DXR, MDR cells. N-Acetyl-L-leucyl-L-leucyl-norleucinal was effluxed by Pgp, but N-benzyloxycarbonyl-L-leucyl-L-leucinal (zLLal), an inhibitor of calpain, retarded the degradation of Pgp leading to accumulation of the molecule largely at the cell surface membrane. Treatment with brefeldin A did not obstruct the zLLal-induced Pgp accumulation. NH4Cl increased the cytoplasmic Pgp level, with a slight to significant decrease at the cell surface membrane. Ubiquitin-ELISA and western blot analysis confirmed that the Pgp molecule, which accumulated mainly at the cell surface, was ubiquitinated. However, lactacystin did not show any accumulation of Pgp in either the cytoplasm or the cell surface membrane, suggesting that the proteasome did not participate in the phenomenon. Additionally, the Pgp was limitedly proteolyzed by calpain into two 98 kDa and 69 kDa, fragments within one minute. Despite the increased accumulation of Pgp at the cell surface after treatment with calpain inhibitor, the cytoplasmic doxorubicin level of the cells treated with a calpain inhibitor was higher than that of non-treated cells and approached that of parental cells. These results indicated that calpain involved Pgp turnover and that calpain inhibition induced ubiquitinated Pgp-accumulation mainly at the cell surface membrane with a reduction in its own functions suggesting that the modulation of Pgp-turnover involves MDR-reversal by another approach.
authors.affiliation: Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ohkawa
authors.forename: T
authors.initials: T
authors.lastname: Asakura
authors.forename: K
authors.initials: K
authors.lastname: Takada
authors.forename: T
authors.initials: T
authors.lastname: Sawai
authors.forename: Y
authors.initials: Y
authors.lastname: Hashizume
authors.forename: Y
authors.initials: Y
authors.lastname: Okawa
authors.forename: N
authors.initials: N
authors.lastname: Yanaihara
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism*; D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D004317:Doxorubicin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D020014:K562 Cells; D007930:Leucine / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D006023:Glycoproteins; D007976:Leupeptins; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D014452:Ubiquitins; C400241:benzyloxycarbonylleucyl-leucine aldehyde; C039304:calpain inhibitors; D000643:Ammonium Chloride; C063261:acetylleucyl-leucyl-norleucinal; C067713:lactacystin; D020126:Brefeldin A; D004317:Doxorubicin; D002154:Calpain; D007930:Leucine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine