Toward localization controllable proteins: subcellular targeting to the nucleus, cytoskeleton, and proteasome

The control of subcellular protein localization to alter function offers great potential for treatment of diseases caused by mislocalization. Further, inducing mislocalization may assist in the determination of a protein's function. To this end, three localization-controllable constructs have been created. A central feature among them is their ability to respond only to an externally administered ligand, which is mediated through the incorporation of a steroid hormone ligand-binding domain. Controlled cytoplasm-to-nucleus translocation can be accomplished through the "protein switch," which was optimized in this work to produce a significant shift in localization from the cytoplasm to the nucleus upon ligand induction. The glucocorticoid receptor ligand-binding domain confers ligand-responsiveness, and nuclear export and nuclear localization signals help shift the balance of localization. Controlled targeting of proteins to the cytoskeleton can be accomplished through the isolation of the estrogen receptor ligand-binding domain. When this domain binds the antagonist fulvestrant, it forms insoluble protein aggregates that associate with the cytoskeleton. A protein fused to the ligand-binding domain will get sequestered in the cytoskeleton, and any cellular activity it had is lost. Controlled targeting of a protein to the ubiquitin-proteasome pathway can be achieved by regulating the access of the tumor suppressor p53 to the nucleus, where it binds the protein responsible for its ubiquitination and proteasomal degradation. When p53 was fused to the protein switch, it remained cytoplasmic before ligand iv addition, but after ligand it translocated to the nucleus and subsequently sent to the proteasome. This technology has the potential to target other intracellular proteins to the proteasome if a binding domain is incorporated. This dissertation focuses on the creation and characterization of these localization-controllable constructs so that they may be used in future applications of protein localization control.