Mechanism of specific nuclear transport of adriamycin: the mode of nuclear translocation of adriamycin-proteasome complex.
Adriamycin (ADM), an anthracycline anticancer agent, is selectively stored in the nuclei of a variety of proliferating cells, but the precise mechanism of specific nuclear transport of ADM is not well known. Recently, we demonstrated that ADM shows high binding affinity to the cytoplasmic proteasomes of L1210 mouse leukemia cells and that taken up ADM by the cells selectively binds to proteasomes. Nuclear targeting of proteasome in proliferating cells may be mediated by the nuclear localization signals that are found in several of the alpha-type subunits of the 20S proteasome. To confirm nuclear transport of the ADM-proteasome complex, we synthesized a photoactive ADM analogue, N-(p-azidohenzoyl)-ADM, and generated a photoaffinity-labeled proteasome complex. The 26S proteasome purified from the cytosol of L1210 cells had a high affinity to N-(p-azidobenzoyl)-ADM. SDS-PAGE analysis of the photoaffinity-labeled proteasome showed that low molecular weight bands (approximately 21-31 kDa) of 20S proteasome had the highest photoaffinity. The photoaffinity-labeled proteasome was distributed in the cytoplasm and nuclei of digitonin-permeabilized L1210 and B-16 mouse melanoma cells in the presence of the cytosolic fraction and ATP. The rate of nuclear translocation of the proteasome was low in the absence of ATP. These results suggest that the proteasome is a specific translocator of ADM from the cytoplasm to the nucleus and that 20S proteasome components are the dominant ADM-binding sites. The nuclear transport of ADM-proteasome complex is regulated by an ATP-dependent nuclear pore-mediated mechanism.
Kiyomiya
K
K
Department of Toxicology, School of Veterinary Medicine, Osaka Prefecture University, Japan. kiyomiya@vet.osakafu-u.ac.jp
Matsuo
S
S
Kurebe
M
M
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004317:Doxorubicin / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007939:Leukemia L1210 / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex
D000345:Affinity Labels; D000903:Antibiotics, Antineoplastic; D009097:Multienzyme Complexes; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004072:Digitonin