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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for all Figures.
Article and author information
Author details
Funding
National Cancer Institute (F31CA265066)
- Antonio Cuevas-Navarro
National Cancer Institute (R35CA197709)
- Frank McCormick
National Cancer Institute (R00CA245122)
- Pau Castel
DOD CDMRP Neurofibromatosis Research Program (W81XWH-20-1-0391)
- Pau Castel
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#AN165444 and #AN179937) of the University of California San Francisco.
Copyright
© 2022, Cuevas-Navarro et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,914
- views
-
- 297
- downloads
-
- 16
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cancer Biology
TAK1 is a serine/threonine protein kinase that is a key regulator in a wide variety of cellular processes. However, the functions and mechanisms involved in cancer metastasis are still not well understood. Here, we found that TAK1 knockdown promoted esophageal squamous cancer carcinoma (ESCC) migration and invasion, whereas TAK1 overexpression resulted in the opposite outcome. These in vitro findings were recapitulated in vivo in a xenograft metastatic mouse model. Mechanistically, co-immunoprecipitation and mass spectrometry demonstrated that TAK1 interacted with phospholipase C epsilon 1 (PLCE1) and phosphorylated PLCE1 at serine 1060 (S1060). Functional studies revealed that phosphorylation at S1060 in PLCE1 resulted in decreased enzyme activity, leading to the repression of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. As a result, the degradation products of PIP2 including diacylglycerol (DAG) and inositol IP3 were reduced, which thereby suppressed signal transduction in the axis of PKC/GSK-3β/β-Catenin. Consequently, expression of cancer metastasis-related genes was impeded by TAK1. Overall, our data indicate that TAK1 plays a negative role in ESCC metastasis, which depends on the TAK1-induced phosphorylation of PLCE1 at S1060.
-
- Cancer Biology
- Cell Biology
Cell crowding is a common microenvironmental factor influencing various disease processes, but its role in promoting cell invasiveness remains unclear. This study investigates the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction in ductal carcinoma in situ (DCIS). Crowding specifically enhanced invasiveness in high-grade DCIS cells through significant volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry revealed that crowding selectively relocated ion channels, including TRPV4, to the plasma membrane in high-grade DCIS cells. TRPV4 inhibition triggered by crowding decreased intracellular calcium levels, reduced cell volume, and increased invasion and motility. During this process, TRPV4 membrane relocation primed the channel for later activation, compensating for calcium loss. Analyses of patient-derived breast cancer tissues confirmed that plasma membrane-associated TRPV4 is specific to high-grade DCIS and indicates the presence of a pro-invasive cell volume reduction mechanotransduction pathway. Hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked crowding-induced effects, while TRPV4 activation reversed them. Silencing TRPV4 diminished mechanotransduction in high-grade DCIS cells, reducing calcium depletion, volume reduction, and motility. This study uncovers a novel pro-invasive mechanotransduction pathway driven by cell crowding and identifies TRPV4 as a potential biomarker for predicting invasion risk in DCIS patients.