The importin β binding domain as a master regulator of nucleocytoplasmic transport

Specific and efficient recognition of import cargoes is essential to ensure nucleocytoplasmic transport. To this end, the prototypical karyopherin importin β associates with import cargoes directly or, more commonly, through import adaptors, such as importin α and snurportin. Adaptor proteins bind the nuclear localization sequence (NLS) of import cargoes while recruiting importin β via an N-terminal importin β binding (IBB) domain. The use of adaptors greatly expands and amplifies the repertoire of cellular cargoes that importin β can efficiently import into the cell nucleus and allows for fine regulation of nuclear import. Accordingly, the IBB domain is a dedicated NLS, unique to adaptor proteins that functions as a molecular liaison between importin β and import cargoes. This review provides an overview of the molecular role played by the IBB domain in orchestrating nucleocytoplasmic transport. Recent work has determined that the IBB domain has specialized functions at every step of the import and export pathway. Unexpectedly, this stretch of ~ 40 amino acids plays an essential role in regulating processes such as formation of the import complex, docking and translocation through the nuclear pore complex (NPC), release of import cargoes into the cell nucleus and finally recycling of import adaptors and importin β into the cytoplasm. Thus, the IBB domain is a master regulator of nucleocytoplasmic transport, whose complex molecular function is only recently beginning to emerge. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.     

Exchange of histidine spacing between Sp1 and GLI zinc fingers: distinct effect of histidine spacing-linker region on DNA binding

In the DNA recognition mode of C(2)H(2)-type zinc fingers, the finger-finger connection region, consisting of the histidine spacing (HX(3-5)H) and linker, would be important for determining the orientation of the zinc finger domains. To clarify the influence of spacing between two ligand histidines in the DNA binding, we exchanged the histidine spacing between Sp1 and GLI zinc fingers, which have an HX(3)H-TGEKK linker (typical) and an HX(4)H-SNEKP linker (atypical), respectively. A significant decrease in the DNA binding affinity and specificity is found in Sp1-type peptides, whereas GLI-type peptides show a mild reduction. To evaluate the effect of the linker characteristics, we further designed Sp1-type mutants with an SNEKP linker. As a result, the significant effect of the histidine spacing in Sp1-type peptides was reduced. These results demonstrate that (1) the histidine spacing significantly affects the DNA binding of zinc finger proteins and (2) the histidine spacing and the following linker regions are one effective target for regulating the DNA recognition mode of zinc finger proteins.