IDENTIFICATION AND CHARACTERIZATION OF A NOVEL PROTEIN INTERACTING WITH RAL-BINDING PROTEIN-1, A PUTATIVE EFFECTOR PROTEIN OF RAL

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral a nd exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts wi th RalBP1 by yeast two-hybrid screening and designated it POB1 (partne r of RalBP1), POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis, POB1 int eracted with BalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction, The binding domain of RalBP1 to POB 1 was distinct from its binding domain to Ral. Ral and POB1 simultaneo usly interacted with RalBP1 in COS cells. The binding of POB1 to RalBP 1 did not affect the GTPase activating activity of RalBP1, Furthermore , POB1 bound to Grb2 but not to Nck or Crk, POB1 was tyrosine-phosphor ylated in COS cells upon stimulation with EGF and made a complex with EGF receptor, These results suggest that RalBP1 makes a complex with P OB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3) containing protein, and Ral.