CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells.
Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkyl-phospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosine-sensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient) cells, but was only widely distributed in the early and late endosomes in KB cells. Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi. However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain.
Francisco
F
Muñoz-Martínez
Cristina
C
Torres
Santiago
S
Castanys
Francisco
F
Gamarro
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D049294:Phospholipid Transfer Proteins / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism
D000970:Antineoplastic Agents; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; C526978:TMEM30a protein, human; D010767:Phosphorylcholine; C105905:perifosine