Pinin nuclear and centrosomal localization and implications in the regulation of epitheliogenesis.

摘要

Epithelial cells associate with their neighbors and with the underlying extracellular matrix (ECM) to form a sheet of cells termed an epithelium. Epithelia protect the underlying tissues, and establish gradients necessary for transport of molecules into and out of the body. To function properly, epithelial cells must faithfully establish structural and functional associations with neighboring cells and tissues. This dynamic process of forming and maintaining the epithelium is termed epitheliogenesis. Epitheliogenesis relies on the appropriate response of epithelial cells to cues from neighboring cells and the surrounding tissues. Our lab has previously characterized a protein termed Pinin (Pnn), which appears to be involved in regulation of adhesion, migration, and proliferation of epithelial cells. An intriguing aspect of Pnn is its localization to regions of the cell where it could play a direct part in regulation of epitheliogenesis. Because of the apparent ability of Pnn protein to influence central epitheliogenic processes, focused studies into the localization of Pnn were conducted.; Pnn association with the nucleus and the centrosome was characterized to test the hypothesis that Pnn localization to theses organelles affected the epithelial qualities of cells. Using a battery of Pnn-GFP fusion proteins, it was determined that Pnn contained two import-active domains spanning 1-88 and 683-717, and two domains 218-285 and 558-638 that were able to target proteins to nuclear speckles. These domains acted independently and in concert to localize Pnn within nuclei. MDCK cells stably transfected with the Pnn nuclear mislocalizing mutant GFP 119-717 demonstrated weakened intercellular adhesion, cytoplasmic spreading, and nuclear enlargement and pleiomorphism. Previous studies also revealed a novel localization for Pnn at centrosomes. Consistent with Pnn centrosomal localization, interaction of Pnn (domain 142-218) with the centrosomal and golgi-related AKAP450 protein (domain 2490-2657) was verified in vitro. Together these data describe the localization of Pnn to nuclei and to centrosomes. The implications of these associations in regulation of epitheliogenesis are discussed. These studies have opened new avenues for investigation of Pnn function in the regulation of epitheliogenesis.