RAC1 ^P29S is a spontaneously activating cancer-associated GTPase

Abstract

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1 P29S ), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1 P29S protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1 F28L-a known spontaneously activated RAC1 mutant-and RAC1P29S are self-activated in distinct fashions. Moreover, the mechanism of RAC1 P29S and RAC1 F28L activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1 P29Sgain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity.cancer | cell signaling | x-ray crystallography | cytoskeleton | GEF-independent GTPase exchange R AC1, the Ras-related small GTPase belonging to the Rho family, functions as a binary molecular switch cycling between an inactive GDP-bound "OFF" state and an active GTP-bound "ON" state (1). Its activity is responsible for the regulation of diverse cellular behaviors including NADPH oxidase activation, formation of cortical actin-containing membrane ruffles and lamellipodia, and induction of gene expression programs (2). Accordingly, these functions are tightly controlled through RAC1 lipidation, subcellular localization, protein expression levels, and Rho GDP-dissociation inhibitor (Rho GDI) interactions. In addition, as a GTPase, RAC1 is turned ON by guanine nucleotide exchange factors (GEFs) and is turned OFF by GTPase activating proteins (GAPs) that facilitate GDP/GTP nucleotide exchange and GTP hydrolysis, respectively. Once this regulation is compromised, RAC1 activity is implicated in various steps of oncogenesis including initiation, progression, invasion, and metastasis (3, 4).In contrast to Ras, RAC1 has rarely been identified as significantly mutated in cancer. Instead, overexpression of RAC1 has been reported in colorectal, pancreatic, breast, and testicular cancers and in various leukemias (5-7). Additionally, a self-activating splice variant of RAC1, RAC1b, was shown to be overexpressed in breast cancer and lung cancer and is thought to mediate the epithelial-mesenchymal transition in lung epithelial cells (8-10). Furthermore, aberrant activation of upstream regulators of RAC1, particularly in the DBL family of GEFs specific for RAC1 (e.g., TIAM1, PREX1, and ECT2), have been implicated in various cancers (11). Although increased GDP→GTP nucleotide exchange in RAC1 (dependent or independent of GEFs) is...

The involvement of mutant Rac1 in the formation of invadopodia in cultured melanoma cells

“…Notably, Rac1 activity levels were very low in the wild-type Rac1 melanoma lines, A375 and 31T (data not shown). These results suggest that the zygosity of RAC1-P29S correlates with RAC1 activation in the cells, strengthening the claim that the mutant encodes for an active RAC1, as previously shown [57]. …”

“…RAC1-P29S was shown to bind more GTP, as well as downstream effectors such as PAK1 and MLK3 [53,54]. It also induces ERK phosphorylation, cell proliferation, membrane ruffling, and transwell migration in normal cells [54,57]. The general mode of action of Rac1, and its P29S mutant, are shown in Fig.…”

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

“…We propose that the GEF domain in PREX2 truncating mutants is activated because of the removal of the autoinhibitory C terminus, as shown for other GEFs including the highly similar PREX1 (24,25,46). Interestingly, Rac1 is recurrently mutated and constitutively activated in cutaneous melanoma (47,48). Hence, overactivation of Rac1 signaling pathway either through direct mutational activation or through its upstream regulators (e.g., PREX2) appears to be an emerging theme in melanoma biology.…”

The role of small GTPase Rac1 in stress signaling

“…Rac1 is also important in various oncogenesis pathways including initiation, progression, invasion and metastasis (Davis et al 2013) and stress sensing (Han et al 2001). The Insert region is essential for mitogenesis and apoptosis.…”

“…Rac1 GEFs have recently been reported to have important roles in cancer (Wertheimer et al 2012;Davis et al 2013). It has been shown that Trp56, which is present between the two switch regions, is the necessary and critical amino acid of Rac1; hence targeting the Trp56 site by an interfering compound can affect the Rac1 activity (Gao et al 2001).…”