Highly homologous proteins exert opposite biological activities by using different interaction interfaces

Amir, Anat; Rosmalen, Martijn van; Mayer, Guy; Lebendiker, Mario; Danieli, Tsafi; Friedler, Assaf

doi:10.1038/srep11629

Cited by 10 publications

(19 citation statements)

References 56 publications

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“…ASPP2 was reported to interact via its N-terminal ubiquitin-like (Ubl) domain with GTP-H-ras [ 27 ], and increase ERK phosphorylation. Phosphorylation of Ser826 in the proline-rich domain of ASPP2 by activated ERK induces ASPP2 translocation to the nucleus [ 3 ], where it interacts via its C-terminal domain with p53 to effect apoptosis [ 28 – 30 ]. It also mediates Ras-induced senescence by inhibiting autophagy in a p53-independent fashion [ 31 ].…”

Section: Introductionmentioning

confidence: 99%

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

et al. 2016

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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.

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Section: Introductionmentioning

confidence: 99%

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

et al. 2016

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“…This is in agreement with our previous findings that the N-terminus of iASPP is required for its cytoplasmic localization [ 20 ] and that phosphorylation of the N-terminal Ser84 by cyclin B1/CDK1 on iASPP disrupts iASPP dimerization [ 11 ] resulting in the exposure of the recently identified RaDAR code located at its C-terminal Ankyrin repeats, which enables its nuclear entry via the RaDAR nuclear import pathway[ 21 ]. Recently, the precise intermolecular interaction of iASPP has also been dissected by Iosub-Amir et al [ 40 ], where they show that the N-terminal iASPP peptides 60-74 and 540-562 have the strongest intermolecular interactions with the C-terminal ankyrin-repeat and SH3 domain. Our work is in keeping with this observation as caspase cleavage would remove the 60-74 residues of N-terminal iASPP important for dimerization and cytoplasmic retention, thereby facilitating its nuclear entry.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, structural and biological data from Iosub-Amir et al provide an alternative explanation for the opposite biological outcomes produced by the ASPP family of proteins [ 40 ]. The intermolecular interaction of iASPP (anti-apoptotic) and ASPP2 (pro-apoptotic) are both mediated by their conserved proline-rich and ankyrin-SH3 domain.…”

Section: Discussionmentioning

confidence: 99%

“…The intermolecular interaction of iASPP (anti-apoptotic) and ASPP2 (pro-apoptotic) are both mediated by their conserved proline-rich and ankyrin-SH3 domain. However, the precise binding sites are different, which suggests that the correct intermolecular interaction is critical for the apoptosis related biological activity of the ASPP proteins [ 40 ]. Furthermore, the cleavage of iASPP in the N-terminal proline-rich domain disrupts this intermolecular interaction with the ankyrin-SH3 domain and thus may affect its original anti-apoptotic biological activity.…”

Section: Discussionmentioning

confidence: 99%

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Caspase cleavage of iASPP potentiates its ability to inhibit p53 and NF-κB

Wang

et al. 2015

Oncotarget

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An intriguing biological question relating to cell signaling is how the inflammatory mediator NF-kB and the tumour suppressor protein p53 can be induced by similar triggers, like DNA damage or infection, yet have seemingly opposing or sometimes cooperative biological functions. For example, the NF-κB subunit RelA/p65 has been shown to inhibit apoptosis, whereas p53 induces apoptosis. One potential explanation may be their co-regulation by common cellular factors: inhibitor of Apoptosis Stimulating p53 Protein (iASPP) is one such common regulator of both RelA/p65 and p53. Here we show that iASPP is a novel substrate of caspases in response to apoptotic stimuli. Caspase cleaves the N-terminal region of iASPP at SSLD294 resulting in a prominent 80kDa fragment of iASPP. This caspase cleavage site is conserved in various species from zebrafish to Homo sapiens. The 80kDa fragment of iASPP translocates from the cytoplasm to the nucleus via the RaDAR nuclear import pathway, independent of p53. The 80kDa iASPP fragment can bind and inhibit p53 or RelA/p65 more efficiently than full-length iASPP. Overall, these data reveal a potential novel regulation of p53 and RelA/p65 activities in response to apoptotic stimuli.

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“…5,14,15 iASPP interacts with numerous apoptosis-related proteins such as the p53 protein family14 and NF-κB,16,17 mainly through its Ank–SH 3 C-terminal domains. We previously showed that the important residues for iASPP interactions with other proteins are iASPP 739–753 and iASPP 764–778 15. Recently it was shown that the Pro domain of iASPP binds the proteins Keap1 and CEP55 18,19.…”

Section: Introductionmentioning

confidence: 99%