Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to asNck-,Ash- andphospholipase Cγ-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck. A NAP4 fusion peptide exhibited an affinity for Nck, Ash and phospholipase Cγ in whole cell lysates. NAP4 also had an SH2 domain, which could bind to activated EGF receptor. These intermolecular interactions imply the intricacy of Nck-mediated signaling around the receptor protein-tyrosine kinases. In addition, NAP4 bore a putative nuclear localization signal and a Q-run/P-run composite, both characteristic of nuclear proteins, and might therefore relate to the presence of Nck in the cellular nucleus.