¹Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, Al Ain 17666, United Arab Emirates

²Department of Pharmacology, RW Johnson Medical School, University of Medicine & Dentistry of New Jersey, Piscataway, New Jersey, NJ 08854, USA

³Ludwig Institute for Cancer Research, Melbourne 3050, Australia

Correspondence to: H Maruta, Ludwig Institute for Cancer Research, Melbourne 3050, Australia

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 could 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 not a specific inhibitor that inactivates only CDC42. Thus, we have taken the minimum CDC42-binding domain (residues 504 - 545, called ACK42) of the Tyr-kinase ACK-1 that binds only CDC42 in the GTP-bound form, and thereby blocking the interactions of CDC42-GTP with its downstream effectors such as ACKs, PAKs and N-WASP. First of all, using the ACK42-GST fusion protein as a specific ligand for the GTP-CDC42 complex, we have revealed that CDC42 is activated 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 (Epidermal Growth Factor) which activate the endogenous normal Ras, providing the first direct evidence that CDC42 acts downstream of Ras and NGF/EGF. Furthermore, over-expression of ACK42 completely reversed Ras-induced malignant phenotypes such as focus formation and anchorage/serum-independent growth of the fibroblasts, and a cell-permeable derivative of ACK42 called WR-ACK42 strongly inhibited the growth of Ras transformants, with little effect on the parental normal cell growth, and also abolished Ras-induced filopodium/microspike formation of the fibroblasts which is CDC42-dependent. These observations unambiguously proved for the first time that the RAS-induced activation of CDC42 is indeed essential for Ras to transform the fibroblasts, and furthermore suggest that ACK42 or its peptidomimetics are potentially useful for genotherapy or chemotherapy of Ras-associated cancer.

CDC42; ACK; NGF; neurite outgrowth; Ras transformation; tumor suppressor

ACK42, CDC42-binding fragment of ACK-1; AGE, agarose gel electrophoresis; BB2R, bradykinin B2 receptor; FCS, fetal calf serum; GAP, GTPase activating protein; GDSs, GDP-dissociation stimulators; GST, glutathione S-transferase; IPTG, isopropyl-beta-galactopyranoside; NGF, nerve growth factor; NOG, neurite outgrowth; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecylsulphate; WR-ACK42, cell-permeable ACK42 derivative