title: Ki-1/57 interacts with RACK1 and is a substrate for the phosphorylation by phorbol 12-myristate 13-acetate-activated protein kinase C.
abstract: Ki-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein that is phosphorylated on serine and threonine residues. When isolated from the Hodgkin's lymphoma analogous cell line L540 Ki-1/57 co-immunoprecipitated with a Thr/Ser protein kinase activity. It has been also found to interact with hyaluronic acid and has therefore been termed intracellular IHABP4 (hyaluronan-binding protein 4). Recent studies demonstrated, however, that Ki-1/57 engages in specific interaction with the chromo-helicase-DNA-binding domain protein 3, a nuclear protein involved in chromatin remodeling and transcription regulation. We used the yeast two-hybrid system to find proteins interacting with Ki-1/57 and identified the adaptor protein RACK1 (receptor of activated kinase 1). Next, we confirmed this interaction in vitro and in vivo, performed detailed mapping studies of the interaction sites of Ki-1/57 and RACK-1, and demonstrated that Ki-1/57 also co-precipitates with protein kinase C (PKC) when isolated from phorbol 12-myristate 13-acetate (PMA)-activated L540 tumor cells and is a substrate for PKC phosphorylation in vitro and in vivo. Interestingly, the interaction of Ki-1/57 with RACK1 is abolished upon activation of L540 cells with PMA, which results in the phosphorylation of Ki-1/57 and its exit from the nucleus. Taken together, our data suggest that Ki-1/57 forms a stable complex with RACK-1 in unstimulated cells and upon PMA stimulation gets phosphorylated on threonine residues located at its extreme C terminus. These events associate Ki-1/57 with the RACK1/PKC pathway and may be important for the regulation of its cellular functions.
authors.affiliation: Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas, SP, Brasil.
authors.forename: Flávia C
authors.initials: FC
authors.lastname: Nery
authors.forename: Dario O
authors.initials: DO
authors.lastname: Passos
authors.forename: Vera S
authors.initials: VS
authors.lastname: Garcia
authors.forename: Jörg
authors.initials: J
authors.lastname: Kobarg
mesh_terms: D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D017730:Ki-1 Antigen / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D017730:Ki-1 Antigen; D010455:Peptides; D000075370:Receptors for Activated C Kinase; C070301:peptide I; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate