ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

Abstract

Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner

“…In agreement with the nanoclustering changes (Figure 1C , 3A ), Gal-1 overexpression and FKBP12 knockdown led to a similar increase in pErk and pS6K1 levels in H-rasG12V transfected HEK cells (Figure 3E ). However, as observed by others and us [ 25 , 32 ], high levels of Gal-1 decreased pAkt, while FKBP12 knockdown increased it (Figure 3E ). No significant changes of the above phosphoproteins were observed when Gal-1 was knocked down or FKBP12 was overexpressed (Figure 3E ).…”

“…We therefore tested, whether rapalogs could phenocopy the observations made with CHX. Given the increasing evidence that H-ras- and K-ras-isoforms have distinct, if not partly antagonistic functions [ 12 , 14 , 24 ], we employed a FRET-assay, which analyses the densely packed, active Ras proteins in isoform-specific signaling nanocluster in the membrane [ 25 , 26 ] (Figure 1A , 1B ). Treatment of HEK293-EBNA (hereafter HEK) cells with rapalogs increased specifically H-rasG12V nanoclustering-FRET to a similar level as the well-established nanocluster scaffold Gal-1 [ 19 ] (Figure 1C , 1D ).…”

Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1

“…Importantly, dynamic nanoclustering of Ras into distinct di- or oligomeric signalling complexes, correlates with effector recruitment and MAPK-signalling output 33 , 34 . Hence, nanoclustering can be modulated to alter Ras-signalling output, such as by modifying membrane lipid organisation 30 , 35 or changing the abundance of nanocluster scaffolding/modulator proteins, such as galectin-1 or apoptosis-stimulating of p53 protein 2 (ASPP2) 33 , 36 .…”

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus

“…Because we found alteration in both PKA and Cdc42 pathways upon IRD truncation, these specific interactions in A. fumigatus may require the IRD. Related to this hypothesis, the IRD might interact with scaffolding proteins important for efficient signaling through various networks (45, 46). Alternatively, the IRD may be directly involved in binding to entirely novel proteins that play important roles in fungal Ras signaling fidelity.…”