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Nuclear Transport and Regulation of the Tumor Suppressor LKB1

Dorfman, Julia
Thesis/Dissertation; Online
Dorfman, Julia
Macara, Ian
Pemberton, Lucy
The nucleus is the defining organelle of the eukaryotic cell. The nuclear envelope separates the nuclear and cytoplasmic compartments of the cell. Nuclear transport occurs through the nuclear pore complexes (NPCs). NPCs are large protein assemblies with a calculated molecular mass of 60 MDa in vertebrates. These structures are assembled from ~30 distinct proteins called nucleoporins (Nups), which are present at the NPC in multiple copies. The NPC was described as a passive sorting device that allows translocation of receptorcargo complexes while excluding other macromolecules. The selectivity of the nuclear pore is based on the ability of transport receptors to interact with unstructured FG-repeat Nups that line the central channel of the NPC. How FGrepeat Nups establish a permeability gradient is not well understood. We examined biophysical properties of FG-repeat Nups in order to test the prevailing models of the nuclear transport. We showed that transport receptors and FGdomains of the Nups lining the central channel are not hydrophobic, in contrast to the premises of selective phase model of nuclear transport. We also provide evidence against intermolecular and intramolecular interactions between FGrepeats. The effect of temperature on passive diffusion of inert molecules and facilitated diffusion of importin suggests that receptor-FG-repeat binding rather than FG-repeat-FG-repeat binding is the dominant factor in nuclear transport. 3 Nuclear transport regulates activity of multiple proteins involved in cell signaling. In fact, multiple tumor suppressors shuttle between the nucleus and the cytoplasm and can be inactivated by mislocalization in cancer. Serine/Threonine kinase LKB1, mutated in Peutz-Jeghers syndrome, is regulated by translocation from the nucleus to the cytoplasm in response to binding to its cofactors STRAD and MO25. We investigated how cofactors of LKB1 control its subcellular localization. We found that while LKB1 is actively imported into the nucleus by importin-/, STRAD and MO25 passively diffuse between the nucleus and the cytoplasm. STRAD facilitates nuclear export of LKB1 by serving as an adaptor between LKB1 and exportins CRM1 and exportin7. STRAD inhibits import of LKB1 by competing with importin for binding to LKB1. MO25 stabilizes the LKB1-STRAD complex but does not facilitate its nucleo-cytoplasmic shuttling. Strikingly, the STRAD isoform, which differs from STRAD in the N-and C-terminal domains that are responsible for interaction with export receptors, does not efficiently relocalize LKB1 from the nucleus to the cytoplasm. These results identify a multi-factored mechanism to control LKB1 localization and suggest that the STRAD-LKB1 complex might possess unique functions in the nucleus. Note: Abstract extracted from PDF text
University of Virginia, Department of Biophysics, PHD, 2008
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