Nuclear localization and degradation of the paired-like:CVC homeobox protein, Vsx-1

Vsx-1 and Vsx-2 are two paired -like homeobox genes that were originally discovered in the regenerating visual pathway of goldfish (Levine and Schechter, 1993; Levine et al., 1994; Levine et al., 1997b) and further studied in the developing zebrafish (Passini et al., 1997; Passini et al., 1998a). During retinal development, the complementary expression of Vsx-1 and Vsx-2 mRNA suggests that Vsx-1 is involved in bipolar cell differentiation whereas Vsx-2 is involved in cell proliferation (Passini et al., 1998a). Such a functional role in retinogenesis suggests that these transcription factors are likely to be temporally and spatially regulated within the cell. Typically, during development transcription factors must be tightly regulated to direct proper development. In this thesis, I report the identification of two pathways by which Vsx-1 protein may be post-translationally regulated; through ubiquitin mediated proteolysis and control of nuclear import. To understand the developmental regulation of Vsx-1 we investigated ubiquitination as a possible post-translational mechanism. In vitro , Vsx-1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx-1-Ubn complexes and stabilized unmodified Vsx-1 Additionally, in transiently transfected COS-7 cells, Vsx-1 is ubiquitinated and degraded. Vsx-1 proteins with C-terminal deletions retained the capacity for initial modification by ubiquitin, however lost the capacity for efficient chain elongation. These results show that Vsx-1 is a substrate of the ubiquitin/proteasome pathway and suggest that C-terminal sequences of Vsx-1 are critical for ubiquitin chain elongation. In addition, these results suggest that ubiquitin-dependent proteolysis regulates Vsx-1 during zebrafish retinal development. In additional studies, we used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier, SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. Using a yeast two-hybrid assay, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH2? terminus of the homeodomain (HD). In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a peri-nuclear ring and co-localizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Co-transfection of Vsx-1 with either human or zebrafish Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1 conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a non-enzymatic mechanism that is independent of SUMO-1 conjugation. During retinal development and growth, Ubc9 may be an integral component of the system that determines the cellular localization and function of Vsx-1.