The CDC42-specific inhibitor derived from ACK-1 blocks v-Ha-Ras-induced transformation

Based on the previous experiments with the N17 mutant of CDC42, it has been speculated, but not proved as yet, that CDC42 is required for Ras-induced malignant transformation of fibroblasts. However, since this inhibitor coul d sequester many GDP-dissociation stimulators (GDSs), such as DBL, OST and Tiam-1 which activate not only CDC42, but also Rho or Rac, in fact it is no t a specific inhibitor that inactivates only CDC42, Thus, we have taken the minimum CDC42-binding domain (residues 504-545, called ACK42) of the Tyr-k inase ACK-1 that binds only CDC42 in the GTP-bound form, and thereby blocki ng the interactions of CDC42-GTP with its downstream effecters such as ACKs , PAKs and N-WASP, First of all, using the ACK42-GST fusion protein as a sp ecific ligand for the GTP-CDC42 complex, we have revealed that CDC42 is act ivated by oncogenic Ras mutants such as v-Ha-Ras in NIH3T3 fibroblasts, and similarly in PC12 cells by both NGF (Nerve Growth Factor) and EGF (Epiderm al Growth Factor) which activate the endogenous normal Ras, providing the f irst direct evidence that CDC42 acts downstream of Ras and NGF/EGF. Further more, over-expression of ACK42 completely reversed Ras-induced malignant ph enotypes such as focus formation and anchorage/serum-independent growth of the fibroblasts, and a cell-permeable derivative of ACK42 called WR-ACK42 s trongly inhibited the growth of Ras transformants, with little effect on th e parental normal cell growth, and also abolished Ras-induced filopodium/mi crospike formation of the fibroblasts which is CDC42-dependent. These obser vations unambiguously proved for the first time that the RAS-induced activa tion of CDC42 is indeed essential for Ras to transform the fibroblasts, and furthermore suggest that ACK42 or its peptidomimetics are potentially usef ul for genotherapy or chemotherapy of Ras-associated cancer.