Molecular and functional characterisation of lm06, a pet-lim protein

Resumen

LIM-domain proteins have crucial roles in cell regulation, usually as interactors to structural or regulatory proteins, but little is known about how, where, and why these interactions occur, or what happens when they do not. The PET-LIM family is an emerging subset of LIM proteins (six members in humans), from which the two best-studied members (TES and Prickle) have key roles in cell adhesion, tumour suppression/progression and planar polarity signaling. Prickle3/LMO6 is a novel member of this family, sharing features of both TES and Prickle. A detailed study of Prickle3/LMO6 could reveal unique roles in cell adhesion, polarity and cancer. Starting from DNA sequence alone, we have developed a battery of useful tools to study this novel protein. During the course of this work, we explored new motifs in Prickle3/LMO6 and confirmed their importance through mutational analysis. We found it is a dynamic nucleocytoplasmic shuttling protein, with strongest localisation in cell-cell adhesions (likely adherens junctions) and nuclear envelope. Prickle3/LMO6 is enriched in epithelial tissues, where it may play a role in adhesion and/or differentiation. In vitro experiments using human keratinocytes demonstrate how calcium-induced differentiation dramatically rearranges the subcellular localisation of Prickle3/LMO6, which is translocated from the cytoplasm to zipper-like nascent adhesions. Using TAP-tagging and candidate approaches, we have identified an assortment of binding proteins for the different domains of Prickle3/LMO6. Proteins identified play key roles in mitosis, transcriptional control and protein degradation. In tumour cells, Prickle3/LMO6 overexpression can block anchorage-independent growth and influence proliferation and migration. Prickle3/LMO6 may also activate E-cadherin expression by counteracting the function of a transcriptional repressor complex. RNAi experiments are underway to test our theories about Prickle3/LMO6 function, with further support from knockout mouse ES cells that we have obtained recently. This work represents the first detailed analysis of Prickle3/LMO6, a novel protein with potential key roles in cell adhesion, migration, epithelial differentiation, transcriptional control and cancer. At the same time, it contributes to a better understanding of the complex and dynamic regulation of LIM and PET-LIM proteins at the cellular and molecular level.