RASSF1A Is Part of a Complex Similar to the Drosophila Hippo/Salvador/Lats Tumor-Suppressor Network

Abstract

The Ras Association Domain Family 1A (RASSF1A) gene is one of the most frequently silenced genes in human cancer. RASSF1A has been shown to interact with the proapoptotic kinase MST1. Recent work in Drosophila has led to the discovery of a new tumor-suppressor pathway involving the Drosophila MST1 and MST2 ortholog, Hippo, as well as the Lats/Warts serine/threonine kinase and a protein named Salvador (Sav). Little is known about this pathway in mammalian cells. We report that complexes consisting of RASSF1A, MST2, WW45 (the human ortholog of Sav), and LATS1 exist in human cells. MST2 enhances the RASSF1A-WW45 interaction, which requires the C-terminal SARAH domain of both proteins. Components of this complex are localized at centrosomes and spindle poles from interphase to telophase and at the midbody during cytokinesis. Both RASSF1A and WW45 activate MST2 by promoting MST2 autophosphorylation and LATS1 phosphorylation. Mitosis is delayed in Rassf1a(-/-) mouse embryo fibroblasts and frequently results in cytokinesis failure, similar to what has been observed for LATS1-deficient cells. RASSF1A, MST2, or WW45 can rescue this defect. The complex of RASSF1A, MST2, WW45, and LATS1 consists of several tumor suppressors, is conserved in mammalian cells, and appears to be involved in controlling mitotic exit.

RASSF1A interacts with and activates the mitotic kinase Aurora-A

“…This phosphorylation might modulate RASSF1A's ability to bind to microtubules, as a phosphorylationmimicking mutant of RASSF1A could not interact with microtubules, whereas a phosphorylation-defective mutant could (Rong et al, 2007). However, endogenous RASSF1A localizes predominantly at centrosomes but not microtubules in interphase cells (Guo et al, 2007; Figure 1). As Aurora-A is inactivate in G 1 (Honda et al, 2000;Castro et al, 2002;Taguchi et al, 2002) and expression of RASSF1A is constant throughout the cell cycle (L Liu and GP Pfeifer, unpublished observation), it is very likely that something other than phosphorylation by Aurora-A prevents RASSF1A from binding to microtubules in G 1 .…”

“…Instead RASSF1A may fine-tune cell-cycle progression or its role is redundant with that of other proteins, for example, its closest homologue NORE1A. Cytokinesis failure occurs more frequently in Rassf1a À/À MEFs than in wild-type MEFs (Guo et al, 2007). The mechanistic basis for cytokinesis failure in Rassf1a À/À MEFs is not clear although it is at least in part a consequence of lack of activation of the mitotic exit kinase LATS1 (Guo et al, 2007).…”

“…Cytokinesis failure occurs more frequently in Rassf1a À/À MEFs than in wild-type MEFs (Guo et al, 2007). The mechanistic basis for cytokinesis failure in Rassf1a À/À MEFs is not clear although it is at least in part a consequence of lack of activation of the mitotic exit kinase LATS1 (Guo et al, 2007). As Aurora-B, which is required for cytokinesis (Giet and Glover, 2001;Guse et al, 2005), interacts with RASSF1A (Figure 4b), it is possible that loss of RASSF1A impedes Aurora-B function.…”

“…Endogenous RASSF1A is primarily a centrosomal protein in interphase cells (Guo et al, 2007) and it colocalizes with mitotic spindles in mitosis ( Figure 1a). Using EGFP-tagged deletion fragments of RASSF1A, we determined that the centrosome and spindle pole localization of RASSF1A requires a minimal domain fragment from amino acid D120 to G193 (Figure 1b).…”

The Hippo pathway regulates stem cell proliferation, self-renewal, and differentiation

“…It has been suggested that Sav1 plays a role in the nuclear translocation of Mst1 . In mammalian cells, Mst1/2 are also activated by binding to Ras association domain family (RASSF) proteins (Oh et al, 2006;Guo et al, 2007) possibly due to alteration of Mst1/2 subcellular localization (Khokhlatchev et al, 2002;Praskova et al, 2004). Recently, Mst1/2 were reported to partially co-localize with actin cytoskeleton, disruption of which leads to mild activation of the kinase (Densham et al, 2009).…”