In the quest to identify Ras effector pathways that could provide a clear mechanistic basis for the observed pro-apoptotic activity of the activated GTPase, a major breakthrough came with the identification of the Ras-Association Domain Family (RASSF) of proteins. The latter encompasses 10 members, RASSF1–RASSF10, with additional isoforms identified due to alternative promoter use or splice variations of some of the members (reviewed in ref. 149
). All members of this family contain a Ras-association (RA) domain which, as the name implies, may enable the proteins to directly interact with, and potentially modulate the activity of, different members of the Ras family. However, not all proteins with an RA domain are capable of interacting with Ras (150
). Overexpressed RASSF1 (151
), RASSF2 (152
) and NORE1 (Novel Ras Effector 1, also known as RASSF5,) (29
) have been shown to be Ras effectors, in that they bind preferentially to Ras in its GTP conformation. However, only RASSF2 (153
) and RASSF5 (29
) have been demonstrated to take part in this interaction in an endogenous environment.
RASSF1A is a tumor suppressor that is frequently silenced by promoter methylation in human tumors (154
). Vos et al. (31
) have demonstrated that the direct association of RASSF1A with the Modulator of Apoptosis-1 (MOAP-1) can activate the pro-apoptotic protein, Bax, to induce cell death in 293T cells. The interaction of MOAP-1 and RASSF1A is stimulated by the presence of activated K-Ras, resulting in synergistic enhancement of cell death. The authors demonstrate that K-Ras(V12) stimulates the ability of RASSF1A-MOAP-1 complex to cause translocation of Bax from the cytosol to the mitochondrial membrane, where it can induce apoptosis. When RASSF1 mRNA levels are reduced by siRNA, K-Ras(V12) cannot efficiently activate Bax. A similar effect is seen with the Y40C effector domain mutant of K-Ras, which has impaired ability to induce RASSF1A-MOAP-1 interaction. Interestingly, C65R, a tumor-derived point mutation of RASSF1A, does not interact with MOAP-1 or induce cell death in the presence or absence of activated K-Ras.
RASSF1A has also been shown to interact with the proapoptotic kinase, MST1(30
), which could also lead to cell death. However, it is not known if this interaction occurs in response to Ras activation. Based on other studies showing that the stimulation of MST1 may activate the kinases LATs1 and LATs2, which leads to down-regulation of the anti-apoptotic Bcl-2 (155
), Vos et al. (31
) suggest the interesting possibility that RASSF1A may regulate multiple pathways to induce apoptosis, leading to its importance as a tumor suppressor.
RASSF1C is a shorter product than RASSF1A, resulting from alternative splicing combined with transcription from a distinct promoter of the RASSF1 gene (157
). RASSF1C has been identified as a mediator of Ras-induced apoptosis when transiently co-expressed with Ras(V12) (151
). This appears to occur through activation of the SAPK/JNK pathway (158
). Others have reported that RASSF1C never associates directly with Ras(V12) (159
). Therefore, the physiological relevance and mechanistic details of the interaction of these proteins will require further investigation.
Like RASSF1A, RASSF2 displays characteristics of both a Ras effector (152
) and a tumor suppressor (160
). Endogenous RASSF2 has been shown to bind to the prostate apoptosis response protein 4 (PAR-4) (161
). The latter is an important tumor suppressor in the prostate (162
) and is capable of inducing apoptosis following translocation to the nucleus (163
). Association of PAR-4 with RASSF2 allows nuclear localization of PAR-4 and formation of this complex is enhanced upon expression of activated K-Ras (161
). In the H441 lung carcinoma cell line, which endogenously expresses activated K-Ras, siRNA-mediated knockdown of K-Ras reduced the interaction of PAR-4 with RASSF2 (161
). In addition, knockdown of RASSF2 impaired K-Ras mediated nuclear localization of PAR-4 and TRAIL-induced apoptosis in prostate cancer cells (161
). Taken together, these data suggest that K-Ras induced apoptosis, linked to PAR-4, may be a key regulatory step for tumor suppression in the prostate.
NORE1, also known as RASSF5, shares approximately 60% homology with RASSF1 at the amino acid level (164
). Khokhlatchev et al. (30
) have demonstrated that endogenous NORE1 and the proapoptotic kinase, MST-1, exist as a complex in 293 cells. Activated K-Ras can recruit this complex to the membrane, through its interaction with NORE1, to induce apoptosis. While H- and K-Ras(V12) were both shown to bind to the MST-1-NORE1 complex, only stimulation by K-Ras(V12) resulted in the induction of apoptosis. The authors attribute this functional divergence to the fact that H-Ras(V12) is much more efficient at stimulating the PI 3-Kinase “survival” pathway than K-Ras (66
), suggesting that perhaps this imbalance tips the scales away from induction of apoptosis. Studies utilizing H-Ras constructs with amino acid substitutions in the effector domain have provided support for this concept. The interaction of Ras with NORE1 occurs through the effector region of Ras (29
), and H-Ras harboring either the T35S or Y40C effector mutations is impaired in NORE1 association. However, the E37G Ras mutant remains capable of associating with NORE1 (30
). Interestingly, the E37G mutation impairs the ability of Ras proteins to activate Raf or PI 3-Kinase. The paper by Khokhlatchev et al. (30
) demonstrates that while overexpression of H-Ras(V12) does not induce NORE1-mediated apoptotic cell death in 293 cells, H-Ras(V12,G37) is capable of causing such death, potentially due to the inability of this protein to stimulate the PI 3-Kinase pathway for survival. Conversely, the authors reveal that overexpression of PI 3-Kinase can mute the apoptotic effect of activated K-Ras. Together these results emphasize the overall importance of the balance between PI 3-Kinase activation of pro-survival pathways and NORE1 activation of pro-death pathways (30
Recombinant RASSF4 (165
) and RASSF6 (166
) have been observed to directly interact with activated Ras, and transient overexpression of these proteins leads to apoptosis that is synergistically enhanced by activated Ras. Additionally, overexpressed RASSF6 was shown to co-immunoprecipitate with MOAP and this interaction is enhanced in the presence of activated K-Ras (166
). However, like RASSF1C, further studies on endogenous protein interactions and details of the pathways leading to apoptotic cell death have yet to be reported.
Of the four newer members of the RASSF family, RASSF7–10 (167
), only RASSF9 has been shown to bind to Ras proteins (168
), but little is known about the cellular effects of this interaction. Although the ability of RASSF7 and -8 to associate with Ras has not yet been investigated, it is interesting that both of these proteins have been reported to be required for cell death by necroptosis, a recently recognized form of non-apoptotic cell death (169
Of the 10 known members of the RASSF family of proteins, only the NORE1-MST-1 complex has been shown to directly interact with a Ras protein and induce apoptosis under physiological conditions (30
). Additionally, the interaction of RASSF2 and PAR-4 was shown to be influenced by endogenous levels of activated Ras in lung carcinoma cells (161
). On the other hand, many of the studies described above for the RASSF proteins have been done with overexpressed proteins and further investigations will need to be completed to determine if these proteins can mediate Ras-induced apoptosis in an endogenous cellular environment.