Dynamics of RASSF1A/MOAP-1 Association with Death Receptors

Abstract

RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-␣ stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

Functional importance of RASSF1A microtubule localization and polymorphisms

“…Detailed five-amino acid deletions revealed that the sequences 131 SQAEI and 300 ELHNFL were required for microtubule localization ( Figure 1b). It is interesting that 131 SQAEI is the ataxia telangeictasia mutated (ATM) phosphorylation site on RASSF1A (Agathanggelou et al, 2001) and 300 ELHNFL is just downstream of the site of association with the only known downstream effector of RASSF1A-mediated cell death, the modulator of apoptosis 1 (MOAP-1 at amino acid 311 REEEEHL) (Baksh et al, 2005;Foley et al, 2008). These results are similar to some of the published observations, which suggest that both N-and C-terminal regions of RASSF1A determine microtubule localization.…”

“…Similarly, RASSF1A self association was preserved in the dMT mutant of RASSF1A in a similar way to wild-type self association ( Figure 4a). This self association is required before RASSF1A recruitment to death receptors (Foley et al, 2008). These data suggest that the dMT mutant of RASSF1A still maintained a tertiary structure to allow both self and MOAP-1 association, but not death receptor association.…”

“…Both C65R and A133S significantly lost their ability to associate with both g- (Figure 6c) and a-tubulin (Figure 6d), whereas the E246K polymorphisms maintained the ability to associate with both g-and The C1 domain associates with membrane lipids and contains a zinc finger binding motif for protein-protein associations with death receptors (such as TNF-R1) (Foley et al, 2008). The ATM site is specifically phosphorylated by the DNA damage control kinase, ATM (Hamilton et al, 2009).…”

“…We next explored how RASSF1A polymorphisms may affect the ability to promote cell death. Two key elements seem to govern the initiation of RASSF1A-dependent cell death: the loss of self association (Foley et al, 2008) and the loss of association with 14-3-3 (Ghazaleh et al, 2010). If left unchecked (by either self associations or associations with 14-3-3), RASSF1A may promote uncontrolled cell death or be involved in abnormal growth-control mechanisms.…”

“…In addition, we carried out two more controls for cysteine changes within the C1 domain of RASSF1A, a C98A and C101A/C102A change (Supplementary Figure 3a). C98 is not part of the C1 domain zinc finger of RASSF1A and does not perturb associations with death receptors (Foley et al, 2008) and can effectively inhibit tumor formation (Supplementary Figure 3a). However, a C101A/C102A change was found within the C1 domain zinc finger of RASSF1A and was found to lack the ability to associate with death receptors (Foley et al, 2008).…”