RASSF6; the Putative Tumor Suppressor of the RASSF Family

Abstract

Humans have 10 genes that belong to the Ras association (RA) domain family (RASSF). Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ)-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo) and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1) are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.

Keywords: Hippo pathway; MDM2; Ras; apoptosis; cell cycle; p53; tumor suppressor.

Figure 1

Structures of Caernorhabditis elegans RSF-1, Drosophila melanogaster dRASSF. Homo sapiens RASSF1A, and Homo sapiens RASSF6. C1, phorbol esters/diacylglycerol-binding domain. RA, Ras association domain. SARAH, Salvador/RASSF/Hippo domain. LIM, Zinc-binding domain present in Lin-11, Isl-1, Mec-3. The PDZ-binding motif of RASSF6 is depicted by a red star. The amino acid number of each protein is shown on the right. The RASSF6-interacting proteins are shown on the bottom. The interactions with MST1/2. MAGI1, and MDM2 are demonstrated at the endogenous level (red letters). Ras binds to the RA domain. MST1/2 (mammalian Ste20-like kinase 1/2) interacts with the SARAH domain. MAGI1 (membrane-associated guanylate kinase inverted 1) binds to the PDZ-binding motif. The interacting regions of MDM2 and MOAP1 (modulator apoptosis 1) are not precisely determined.

Figure 2

(A) Interaction of RASSF6 and DLG1. Endogenous RASSF6 was immunoprecipitated from rat liver with anti-RASSF6 antibody. DLG1 was co-immunoprecipitated; (B) FLAG-RASSF6 was expressed in HEK293 cells and immunoprecipitated with anti-FLAG antibody. Endogenous Lin-7 was co-immunoprecipitated; (C) FLAG-RASSF6 was co-expressed with Myc-RASSF1A, RASSF2, RASSF3, RASSF4, Nore1 (for simplicity, described as RASSF5 in this figure), and RASSF6. The immunoprecipitation was performed with anti-Myc antibody. The lower panel was the immunoblotting of the inputs. The upper panel was the immunoblotting of the immunoprecipitates. All Myc-C-RASSF proteins were co-immunoprecipitated with FLAG-RASSF6.

Figure 3

The core structure of the Drosophila Hippo pathway. Hippo and Warts form the kinase cassette. Mats and Salvador function as an activator and a linker to promote the Hippo-mediated activation of Warts. Salvador harbors two WW domains and the SARAH domain. Unphosphorylated Yorkie co-operates with Scalloped to regulate the transcription of cell cycle-promoting and anti-apoptotic genes. When Yorkie is phosphorylated by Warts, Yorkie is recruited from the nucleus to the cytoplasm and undergoes degradation (small entities symbolize degradation). dRASSF blocks the interaction between Hippo and Salvador. Inset: dRASSF suppresses the cell overproliferation phenotype caused by the hippo mutant lacking the SARAH domain but cannot rescue the phenotype caused by the kinase-dead mutant.

Figure 4

RASSF1A functions as an upstream activator for the Hippo pathway. For the precise mechanism of how RASSF1A activates MST2, the readers should refer to the other chapter in this issue. In contrast, RASSF6 works as a partner of MST kinases. Under the normal condition, RASSF6 and MST1/2 form a complex and inhibit each other. Under the condition that the Hippo pathway is activated, RASSF6 and MST1/2 are dissociated. MST1/2 are autophosphorylated (P). RASSF6-mediated apoptosis is concomitantly triggered. Note that, in the case that the machinery underlying RASSF6-mediated apoptosis is impaired, RASSF6 overexpression could lead to oncogenesis through the inhibition of the Hippo pathway.

Figure 5

Under the normal condition, MDM2 degrades RASSF6 and p53. When cells are exposed to stress such as ultraviolet exposure, MDM2 self-ubiquitination is enhanced and p53 is stabilized. RASSF6 also induces apoptosis independently of p53, but the mechanism remains to be clarified. Small entities symbolize degradation of each ubiquitinated protein (Ub-RASSF6, Ub-p53, and Ub-MDM2).