Modulator of Apoptosis 1: A Highly Regulated RASSF1A-Interacting BH3-Like Protein

Abstract

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in both the intrinsic and extrinsic modes of cell death or apoptosis. MOAP-1 is part of the Ras association domain family 1A (RASSF1A)/MOAP-1 pro-apoptotic extrinsic signaling pathway that regulates apoptosis by utilizing death receptors such as tumor necrosis factor α (TNFα) or TNF-related apoptosis-inducing ligand (TRAIL) to inhibit abnormal growth. RASSF1A is a bona fide tumor suppressor gene that is epigenetically silenced by promoter-specific methylation in numerous human cancers. MOAP-1 is a downstream effector of RASSF1A that promotes Bax activation and cell death and is highly regulated during apoptosis. We speculate that MOAP-1 and RASSF1A are important elements of an "apoptotic checkpoint" that directly influences the outcome of cell death. The failure to regulate this pro-apoptotic pathway may result in the appearance of cancer and possibly other disorders. Although loss of RASSF1A expression is frequently observed in human cancers, it is currently unknown if MOAP-1 expression may also be affected during carcinogenesis to result in uncontrolled malignant growth. In this article, we will summarize what is known about the biological role(s) of MOAP-1 and how it functions as a downstream effector to RASSF1A.

Figure 1

Gene structure of human MOAP-1. The entire protein coding sequence of MOAP-1 is contained within exon 3 and is located on the anti-sense strand of chromosome 14. Genbank accession: NM_022151.4. More information can be found at http://www.ncbi.nlm.nih.gov/gene/64112. Numbers below schematic denote the size of the intron or exon.

Figure 2

A comparison of MOAP-1 orthologs. (a) Multiple sequence alignments of MOAP-1 orthologs present in human (h), mouse (m), rat (r) and chimpanzee (c). Sequence alignments were performed using ClustalW2. NCBI reference sequences (mRNA and protein): NM_022151.4 and NP_071434.2 (human); NM_022323.7 and NP_071718.1 (mouse); NM_001013101.1 and NP_001013119.1 (rat); XM_510137.3 and XP_510137 (chimpanzee). (b) Percent amino acid identity between MOAP-1 orthologs calculated based on sequence alignments in (2a). Analysis carried out using ClustalW2.

Figure 3

Tumor inhibiting potential of the RASSF1A/MOAP-1 tumor suppressor pathway. A classical xenograft assay was carried out. Male athymic nude mice were injected subcutaneously with 1 × 10⁶ transiently transfected HCT116 cells mixed with matrigel mix into the right and left flank areas. Tumor volumes were measured until day 35 and plotted. P values for MOAP-1 versus vector (0.019); RASSF1A versus vector (0.0001); MOAP-1 versus RASSF1A (0.02), n = 12–14. Statistical analysis was evaluated by Student's t-test (two-tailed). Protein expression at the time of subcutaneous injection was confirmed by immunoblotting (data not shown). Protein expression in HCT116 cells can be detected up to 10 days post-transfection. However, at the end of experiment, we could not detect protein expression of HA-RASSF1A or Myc-MOAP-1 in the resulting tumors. We argue that the growth properties of HCT116 cells containing the indicated expression constructs were programmed within the first 7–10 days and continued on that program even though expression detection of the indicated genes was not possible. Please refer to [1] for more details on this issue.

Figure 4

A schematic of MOAP-1 with indicated areas of contact with other proteins documented above schematic. Residues empirically determined to be required for protein interactions are indicated below each region.

Figure 5

MOAP-1 cooperates with RASSF1A during death receptor-dependent apoptosis and promotes Bax activation. In response to death receptor stimulation, MOAP-1 is first recruited to the receptor and then followed by RASSF1A association at the MOAP-1/receptor complex. The association of MOAP-1 to RASSF1A promotes a conformational change in MOAP-1 that exposes its BH3-like domain required for Bax association. The subsequent interaction between MOAP-1 and Bax induces a conformational change in Bax that enables its translocation, and insertion into the mitochondrial outer membrane resulting in the release of cytochrome c and other apoptogenic factors, leading to apoptosis.