title: The carboxyl-terminal segment of the adaptor protein ALX directs its nuclear export during T cell activation.
abstract: The adaptor protein ALX acts downstream of CD28 to regulate the interleukin-2 (IL-2) promoter during T cell activation. Whereas ALX is predominantly localized to the cytoplasm, ALX partially resides in the nucleus, and the nuclear pool is rapidly depleted in response to T cell receptor (TCR)/CD28 signaling. Here it is shown that this depletion occurs via nuclear export of ALX, which depends on a leucine-rich nuclear export signal (NES) in its carboxyl segment and on the CRM-1 transport protein. Nuclear import of ALX also depends on its carboxyl-terminal segment. Blocking nuclear export of ALX, either pharmacologically, by leptomycin B, or by site-directed mutation of the ALX NES, impairs CD28-mediated phosphorylation of ALX. Additionally, upon overexpression, the ALX NES mutant was found to be impaired in inhibiting TCR/CD28-induced transcriptional up-regulation of the RE/AP composite element from the IL-2 promoter, whereas a truncated form of ALX that is a potent inhibitor of RE/AP activation was found to reside entirely in the cytoplasm. Together, these results show that ALX exerts its effect on IL-2 up-regulation in the cytoplasm and suggest an intricate relationship between the nuclear localization/export, phosphorylation, and activity of ALX in response to TCR and CD28 signaling.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. shapirov@mail.med.upenn.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shapiro
authors.forename: Yen-Yu
authors.initials: YY
authors.lastname: Chen
authors.forename: Virginia Smith
authors.initials: VS
authors.lastname: Shapiro
mesh_terms: D021581:Active Transport, Cell Nucleus; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D018106:CD28 Antigens / Q000096:biosynthesis; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D028884:Karyopherins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D049790:Nuclear Export Signals; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction; D013045:Species Specificity; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D018909:src Homology Domains; D000097606:Exportin 1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; D000939:Epitopes; D005231:Fatty Acids, Unsaturated; D007376:Interleukin-2; D028884:Karyopherins; D008156:Luciferases; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C442142:HSH2D protein, human; C038753:leptomycin B