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 single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.
Chen
T
T
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, and Department of Oncology, McGill University, Montreal, Quebec, Canada.
Damaj
B B
BB
Herrera
C
C
Lasko
P
P
Richard
S
S
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006367:HeLa Cells; D015801:Helminth Proteins / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011072:Poly U / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D012313:RNA / Q000502:physiology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid
D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D004268:DNA-Binding Proteins; C489561:Dok1 protein, mouse; C074561:GAP-associated protein p62; C094448:GLD-1 protein, C elegans; C053819:GRP33 protein, Artemia salina; D015801:Helminth Proteins; D019476:Insect Proteins; C489559:KHDRBS1 protein, human; C513253:Khdrbs1 protein, mouse; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; C490832:QKI protein, human; C098217:Qk protein, mouse; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011072:Poly U; D012313:RNA; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn