INTERACTION OF NCK-ASSOCIATED PROTEIN-1 WITH ACTIVATED GTP-BINDING PROTEIN RAC

Bacterially expressed glutathione S-transferase fusion proteins contai ning Rad were used to identify binding proteins of this Rho family GTP ase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusivel y with guanosine 5'-[gamma-thio]-triphosphate-bound Rad, not with GDP- bound Rad. The 85 and 110 kDa proteins were identified as the regulato ry and catalytic subunits respectively of phosphatidylinositol 3-kinas e. Several lines of evidence suggested that the 125 kDa protein is ide ntical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibod ies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecip itated with a constitutively active form of Rac expressed in Chinese h amster ovary cells. The observation that complex formation between act ivated Rac and PAK, but not that between Rac and Nap1, could be reprod uced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a po tential candidate for a link that connects Nap1 to Rac. The multimolec ular complex comprising Rac, Nap1 and probably the 140 kDa protein mig ht mediate some of the biological effects transmitted by the multipote nt GTPase.