Sub Cellular Localization of Suppressor of Cytokine Signaling 6 (SOCS6)

Src is a nonreceptor tyrosine kinase that regulates cell proliferation, differentiation and migration.  Deregulated Src causes malignant transformation when overexpressed. Active Src protein is downregulated by the E3 ubiquitin ligase Cullin 5 (Cul5).  Cul5 forms a complex with SOCS (suppressor of cytokine signaling) adaptors to recruit phosphorylated substrates and adds ubiquitin to these substrates thus targeting them for degradation.  These findings maybe important in cancer because Src proteins levels are often increased, while Cul5 and SOCS proteins levels are often decreased in human tumors. Depletion of Cul5 in epithelial cells causes increased levels of Src and the Src substrate, Cas. Cul5-deficient epithelial cells show several Src-dependent transformed phenotypes, including increased migration. The increased migration of Cul5-deficient cells requires Src and Cas. Our lab has identified SOCS6 as the adaptor that binds pY-Cas. SOCS6 also regulates Cas protein levels. This suggests that SOCS6 may recruit pY-Cas to Cul5 for degradation and thereby inhibit cell migration.

The goal of my project is to determine the subcellular localization of SOCS6 in migrating epithelial cells. SOCS6 may bind its substrates in the cytoplasm or in focal adhesions at the leading edge of migrating cells. I have used focal adhesion markers like vinculin and FAK to visualize focal adhesions in migrating cells in a scratch wound assay. I have cloned SOCS6 from human epithelial cells. I will fuse it with a fluorescent tag, express it in MCF10A epithelial cells and use immunofluorescence to visualize its localization in migrating cells.