Augmentation of CTLA-4 expression by wortmannin: involvement of lysosomal sorting properties of CTLA-4.
CTLA-4 (CD152) is transiently induced on the cell surface of activated T cells and expression is limited at a low level. In this study, we investigated the possibility that phosphatidylinositol 3 kinase (PI 3-K) and other related PI kinases associated with the cytoplasmic domain of CTLA-4 are involved in intracellular trafficking and sorting of CTLA-4 protein. Treatment with micromolar concentrations of wortmannin (WN) for >4 h enhanced both cell-surface and intracellular CTLA-4 without affecting its transcriptional activities in a murine mastocytoma cell line transfected with the human CTLA-4 gene and normal activated CD4(+) T cells. However, a more specific PI 3-K inhibitor, LY294002, failed to affect CTLA-4 expression, indicating that the action of WN is independent of conventional PI 3-K activities. WN down-regulated specific association of CTLA-4 with adaptor proteins and its endocytosis. The fact that lysosomotropic agents, ammonium chloride and monensin, enhanced CTLA-4 expression suggests that WN may also block lysosomal sorting and consequent degradation of CTLA-4. Co-localization of CTLA-4 and lysosome-associated membrane protein-1 detected by immunofluorescence microscopy indicates the actual lysosomal sorting of CTLA-4. Our data suggest the existence of WN-sensitive enzymes, which promote lysosomal sorting of CTLA-4. In addition to rapid endocytosis by clathrin-associated adaptor complex, a prompt sorting of CTLA-4 to lysosomes may be one of the regulatory mechanisms for managing CTLA-4 signals in intracellular trafficking pathways.
Oki
S
S
Department of Immunology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-Ku, Tokyo 154-8509, Japan.
Kohsaka
T
T
Azuma
M
M
D000069594:Abatacept; D000643:Ammonium Chloride / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D060908:CTLA-4 Antigen; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D018796:Immunoconjugates; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D002867:Chromones; C556707:Ctla4 protein, mouse; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D009025:Morpholines; D012333:RNA, Messenger; D000643:Ammonium Chloride; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000069594:Abatacept; D008985:Monensin; D000077191:Wortmannin