Maspina (SERPINB5) é um potencial gene supressor de tumor com atividades biológicas pleiotrópicas, incluindo a regulação da proliferação, morte, adesão e migração celular e a expressão gênica e a resposta ao estresse oxidativo. O mecanismo molecular subjacente a sua função é pouco conhecido. Vários estudos sugerem que a localização subcelular de maspina tem um papel essencial na sua função biológica e sua atividade supressora de tumor. A maspina nuclear tem sido associada a um bom prognóstico, enquanto que a sua localização núcleocitoplasmática tem sido correlacionada à progressão tumoral. Portanto, este trabalho teve como objetivo investigar o mecanismo molecular da translocação nuclear de maspina. Assim, identificamos um sinal de localização nuclear (NLS) bipartido na sequência de maspina usando um software de predição de NLS. Deleção do NLS resulta em diminuição dos níveis de maspina nuclear. No entanto, observamos que maspina também é capaz de se difundir ao núcleo em células HeLa permeabilizadas com digitonina. Porém, estudos prévios indicaram que a localização subcelular de maspina é regulada na célula intacta. Assim, para poder distinguir o transporte regulado do passivo, fusionamos a sequência inteira de maspina e do seu NLS a 5GFPs (MaspinFL e 5GFPs-MaspinNLS, respectivamente). Foi observado que o NLS de maspina, mas não a sequência inteira da proteína, foi capaz de translocar a proteína quimérica ao núcleo, o que sugere que a disponibilidade do NLS de maspina pode ser regulada na sua estrutura nativa. Além disso, foi observado que a translocação nuclear de 5GFPs-MaspinNLS foi inibida pela co-transfecção com mutantes de Ran-GTPase, indicando que o processo depende de Ran-GTPase e portanto ocorre ativamente. Não observamos uma interação do NLS de maspina com carioferina alfa 2 (KPNA2) nem uma inibição do transporte nuclear de maspina ao tratar células com importazole, um inibidor da via clássica, o que sugere que maspina transloca para o núcleo de uma forma não convencional. / Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration, gene expression and oxidative stress response. The molecular mechanism underlying maspin function is poorly understood. Several studies suggest that subcellular localization plays an essential role on maspin biological function and tumor suppression activity. Nuclear maspin has been associated with a good prognostic, whereas nucleocytoplasmic localization correlates with tumor progression. The objective of this project was to investigate the mechanism underlying maspin nuclear translocation. We identified a bipartite Nuclear Localization Signal (NLS) in maspin protein sequence using an NLS prediction software. Deletion of maspin NLS leads to decrease in maspin nuclear levels. However, we also observed that maspin diffuses into the nucleus of digitoninpermeabilized cells. Still, previous studies indicated that maspin subcellular localization is regulated in the intact cell. Considering this, in order to distinguish between regulated and passive nuclear transport, maspin NLS sequence and full-length protein sequence were fused to 5GFPs (5GFPs-MaspinNLS and MaspinFL, respectively). We observed that MaspinNLS, but not MaspinFL, was able to drive 5GFPs nuclear translocation, suggesting that the availability of maspin NLS may be regulated in the native maspin structure. Furthermore, 5GFPs-MaspinNLS nuclear translocation was abrogated by mutant Ran-GTPase co-transfection, indicating that this process depends on Ran-GTPase and it occurs actively. An interaction between Maspin NLS and karyopherin alpha 2 (KPNA2) was not detected neither an inhibition of maspin nuclear transport when cells were treated with importazole, an inhibitor of the classic nuclear import pathway. These data suggest that maspin may be translocating to the nucleus in a nonconventional manner.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Signal transduction by transforming growth factor β (TGF-β) cytokines is mediated by an evolutionarily conserved mechanism that depends on the Smad proteins to transduce an extracellular stimulus into the nucleus. In the unstimulated state, Smads spontaneously shuttle across the nuclear envelope and distribute throughout the cell. Upon TGF-β or bone morphogenetic protein (BMP) stimulation, the receptor-activated Smads are phosphorylated, assemble into complexes with Smad4, and become mostly localized in the nucleus. Such signal-induced nuclear translocation of activated Smads is essential for TGF-β–dependent gene regulation that is critical for embryonic development and homeostasis. The molecular machinery responsible for this process, especially how the activated Smads are imported as complexes, is not entirely clear. Thus, I became interested in investigating the molecular requirements for nuclear targeting of Smads upon stimulation. Recently, whole-genome RNAi screening offers a complementary cell-based approach to functionally identify molecules that mediate nuclear accumulation of Smads in response to TGF-β. In the first part of this dissertation, I performed a genome-wide RNAi screen that uncovered the importin moleskin (Msk) required in nuclear import of Dpp-activated MAD. Both genetic and biochemical studies further confirmed this finding. I also investigated Smad interactions with the Msk mammalian orthologues, Importin7 and 8 and validated that Smads are bona fide cargos of Imp7/8. Besides the importin Msk, the screen also uncovered a subset of nucleoporins as required factors in signal-induced nuclear accumulation of MAD. Thus in the second part of this thesis, I focused on how the NPC mediates this Msk-dependent nuclear import of activated MAD. Most of these nucleoporins, including Sec13, Nup75, Nup93 and Nup205, were thought to be structural nucleoporins without known cargo-specific functions. We, however, demonstrated that this subset of nucleoporins was specifically used in the Msk-dependent nuclear import of activated MAD but not the constitutive import of cargos containing a classic nuclear localization signal (cNLS). I also uncovered novel pathway-specific functions of Sec13 and Nup93. Regulation of TGF-β signaling can be achieved not only by modulating Smad nuclear translocation but also by modifying Smad phosphorylation status. Previously we identified a kinase, Misshapen (Msn), that caused the linker phosphorylation of MAD, resulting in negative regulation of Dpp signaling (Drosophila BMP). In the third part of this thesis, I investigated the biological relevance of Msn kinase to Dpp signaling in Drosophila wings. Both over-expression and RNAi studies suggest that Msn is a negative regulator of the Dpp/MAD pathway in vivo. As a whole, my findings delineated two critical requirements for MAD nuclear import: the importin Msk and a unique subset of nucleoporins. For the first time, structural Nups are implicated in the direct involvement of cargo import, providing a unique trans-NPC mechanism.