UC San Diego

Cell polarity is fundamental for the architecture and function of epithelial tissues. Epithelial polarization requires coordination of multiple fundamental cell processes, whose integration in space and time dictates overall epithelial morphogenesis. Loss of epithelial polarity not only impacts organ development and function, but also causes cancers. Here we identify G[alpha]- Interacting Vesicle-associated protein (GIV; a.k.a. Girdin) as a novel substrate of AMPK, whose phosphorylation at a single site (Ser 245) is required for the stabilization of tight junctions (TJs) in polarized epithelial cells. Expression of a nonphosphorylatable GIV mutant S245[right arrow]A made cells highly susceptible to energetic stress or low calcium conditions, triggering early disassembly and impaired reassembly of functional TJs. By contrast, expression of phosphomimetic mutant (S245[right arrow]D) made epithelial cells relatively resistant to energetic stress or low calcium conditions. Phosphorylation of GIV at S245 was also essential for epithelial morphogenesis. Furthermore, we show that an oncogenic mutant (L249[right arrow]P) of GIV impairs AMPK- dependent phosphorylation of GIV at S245 and recapitulated the phenotypes of cells expressing the non- phosphorylatable S245[right arrow]A mutant. More importantly, expression of either S245A or L249P mutants preferentially triggered anchorage-independent growth and transformation, whereas the expression of the constitutively phosphorylated S245D mutant restricted growth to an anchorage-dependent state. Taken together, we conclude that the AMPK-GIV axis stabilizes TJs, and that GIV is a key effector of AMPK at the TJs. This work illuminates a previously unforeseen mechanism by which metabolic stress may affect epithelial polarity, junctional integrity and epithelial morphogenesis via GIV, and how deregulation of this pathway may affect key growth properties of tumor cells