Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of beta 1,4-galactosyltransferase

The beta1,4-galactosyltransferase family contains at least seven unique gen e products, of which beta1,4-galactosyltransferase I (GalT) is the most exh austively studied. GalT exists in the Golgi complex, similar to many other glycosyltransferases, as well as on the cell surface, where it functions as a signaling receptor for extracellular glycoside ligands. When expressed o n the surface, GalT associates with the cytoskeleton and, upon ligand-induc ed aggregation, induces cell-type specific intracellular signal cascades. I n an effort to define the mechanisms by which surface GalT exerts these int racellular effects, we used the yeast two-hybrid system to identify protein s that specifically interact with the GalT cytoplasmic domain. The yeast two-hybrid screen identified two distinct clones (1.12 and 2.52) that showed identity to portions of SSeCKS ((S) under bar rc (S) under bar uppressed (C) under bar (C) under bar inase (S) under bar ubstrate). SSeCKS is a previously defined kinase and cytoskeleton scaffolding protein whose subcellular distribution and functions are remarkably similar to those attr ibuted to GalT. Both SSeCKS and GalT have Been localized to the perinuclear /Golgi region as well as to filopodia/lamellipodia. SSeCKS and GalT have be en implicated in regulating cell growth, actin filament dynamics, and cell spreading. Interestingly, 1.12 and 2.52-GFP constructs were localized to su bcellular domains that correlated with the two purported subcellular distri butions for GalT; 2.52 being confined to the Golgi, whereas 1.12 localized primarily to filopodia, Coimmunoprecipitation assays demonstrate stable bin ding between the GalT cytoplasmic domain and the 1.12 and 2.52 domains of S SeCKS in appropriately transfected cells. Similar assays demonstrate bindin g between the endogenous GalT and SSeCKS proteins also. Coimmunoprecipitati on assays were performed in both directions and produced similar results (i .e. using either anti-GalT domain or anti-SSeCKS domain antibodies as the p recipitating reagent). A functional interaction between the GalT cytoplasmi c domain and SSeCKS was illustrated by the ability of either the 1.12 or 2. 52 SSeCKS domain to restore a normal adhesive phenotype in cells overexpres sing the TL-GFP dominant negative construct. TL-GFP is composed of the GalT cytoplasmic and transmembrane domains fused to GFP, and leads to a loss of cell adhesion on laminin by displacing the endogenous GalT from its cytosk eleton binding sites. This is the first reported interaction between a glyc osyltransferase and a scaffolding protein, and suggests that SSeCKS serve t o integrate the various functions ascribed to the GalT cytoplasmic domain.