Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

Abstract

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of classical RAS GTPases. Here, we have analyzed the phenotypes of Lztr1 loss-of-function mutants in both fruit flies and mice and have demonstrated a biochemical preference for their RIT1 orthologs. Moreover, we show that Lztr1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of Rit1. Overall, our results indicate that, in model organisms, RIT1 orthologs are the preferred substrates of LZTR1.

Ras-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy

“…The copyright holder for this preprint this version posted November 3, 2022. ; https://doi.org/10.1101/2022.11.02.514888 doi: bioRxiv preprint RIT1 membrane trafficking on RAF activation, we expressed N-terminal and C-terminal RIT1 deletion mutants and assessed binding by pulldown. As predicted, deletion of the RIT1 C-terminus (192-219), but not its N-terminus (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), disrupted membrane association, RAF1 binding, and MAPK activation (Fig. 6A and fig.…”

“…Interestingly, preferential RAF1 binding is a property also shared by the classical Ras family members in cells (24). Accumulation of RIT1 through the loss of LZTR1-mediated proteasomal degradation increases MAPK signaling, a defining pathognomonic feature of RASopathies (13,15,41). However, as with other NS germline mutants, pathologic RIT1 signaling is mild and thus compatible with embryonic development (1).…”

“…Accumulation of proteolysis-deficient, pathogenic RIT1 protein recruits RAF kinases to the PM thereby "priming" complete RAF activation by PM resident GTP-loaded Ras. 15 N RBD (black) and 15 N RBD in complex with unlabeled WT-RIT1-GppNHp (blue). (B) Overlay 15 N -1 H HSQC spectra of 15 N RBD (black) and 15 N RBD in complex with unlabeled RIT1 A57G -GppNHp (red).…”

“…The copyright holder for this preprint this version posted November 3, 2022. ; https://doi.org/10.1101/2022.11.02.514888 doi: bioRxiv preprint involves the NS-associated protein LZTR1, which functions as a conserved substrate receptor for Cullin3 E3 ubiquitin ligase complexes (CRL3 LZTR1 ) to promote the ubiquitination and proteasomal degradation of GDP-bound RIT1 (15). NS pathogenic RIT1 and LZTR1 variants disrupt RIT1-LZTR1 binding, resulting in the accumulation of RIT1 protein (13,16) and enhanced MAPK signaling; however, it remains unclear how increased RIT1 protein abundance contributes to MAPK pathway activation.…”

Exploring the Regulation of Cdc42 Stability and Turnover in Yeast

“…One type of regulation impacts Rho GTPase stability, which is mainly controlled by ubiquitination (Deng and Huang, 2014; Guo and Rahmouni, 2019; Majolée et al, 2021; Vanneste et al, 2020; Wei et al, 2013). Indeed, many G-proteins, including RAS (Abe et al, 2020; Bigenzahn et al, 2018; Campbell and Philips, 2021), Ras-like proteins (Cuevas-Navarro et al, 2022), and heterotrimeric G-proteins that function with seven-transmembrane receptors (Dohlman and Campbell, 2019; Wang et al, 2005) are similarly regulated. Rho GTPases are also regulated by other PTMs, including lipid modifications (Michaelson et al, 2001; Navarro-Lérida et al, 2012), phosphorylation (Chang et al, 2011; Cho et al, 2018; Tkachenko et al, 2011), SUMOylation (Yue et al, 2017), and oxidation (Mitchell et al, 2013), which can modulate GTPase localization, activity and function in specific contexts.…”