Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rapt effector. Here we report that, in HEK293T cells. Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for memebrane-association and TNIK activation, whereas all Rapt proteins, including Rap2B, require pahnitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rapt proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rapt proteins. (C) 2008 Elsevier Inc. All rights reserved.