Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Ccn-binding protein rPICK1

TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones fr om a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol . Chem 271, 15034-15044]. The amino acid sequence deduced from clone 5A sho ws 96.9% identity with the murine PICK1, a protein kinase C alpha (PKC alph a)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with th e TIS21 translated in vitro, suggesting a direct physical interaction betwe en these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the intera ction also occurs in mammalian cells. Deletion of the PDZ domain at the N-t erminus of rPICK1 abolishes its interaction with TIS21, A putative carboxyl ate-binding loop required for PICK1 to bind PKCa [Standinger, Lu and Olson (1997) J. Biol. Chem 272, 32019-32024] is within this deleted region. Our r esults suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. T he catalytic activity of PKC towards TIS21 is significantly decreased in th e presence of rPICK1, whereas phosphorylation of histone by PKC is not affe cted, rPICK1 seems to modulate the phosphorylation of TIS21 through specifi c interactions between these two proteins. TIS21 might have a role in PKC-m ediated extracellular signal transduction through its interaction with rPIC K1.