SELF-ASSOCIATION OF THE SINGLE-KH-DOMAIN FAMILY MEMBERS SAM68, GRP33,GLD-1, AND QK1 - ROLE OF THE KH DOMAIN

Sam68 is a member of a growing family of proteins that contain a singl e KH domain embedded in a larger conserved domain of similar to 170 am ino acids, Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other RH domains and contain conserved residues . RH domains are protein motifs that are involved in RNA binding and a re often present in multiple copies. Here we demonstrate by coimmunopr ecipitation studies that Sam68 self-associated and that cellular RNA w as required for the association. Deletion studies demonstrated that th e Sam68 KH domain loops 1 and 4 were required for self-association. Th e Sam68 interaction was also observed in Saccharomyces cerevisiae by t he two-hybrid system, In situ chemical cross-linking studies in mammal ian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 co mplexes bound homopolymeric RNA and the SH3 domains of p59(fyn) and ph ospholipase C gamma 1 in vitro, demonstrating that Sam68 associates wi th RNA and signaling molecules as a multimer. The formation of the Sam 68 complex was inhibited by p59(fyn), suggesting that tyrosine phospho rylation regulates Sam68 oligomerization. Other Sam68 family members i ncluding Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 asso ciated,vith other KH domain proteins such as Bicaudal C. These observa tions indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-prot ein interactions.