The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1

Abstract

Background DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. Methods Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. Results We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. Conclusion These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

DLC3/Cv-c function in testis development in humans and Drosophila: implication for variants of sex development

“…PS microdomains become anchoring points for proteins that promote signal transduction and the stabilization of the junction like Flotillins and PS-binding proteins (Yap et al , 2015). Taking these findings together, we hypothesise a role for the StART domain in the interaction of DLC3 with the temporal PS nanoclusters formed at the AJs, a phenomenon already demonstrated for DLC1 (Sanchez-Solana et al ., 2021). Mutated DLC3-StART domain lacking the ability to respond to lipid-binding with a conformational change, could be incapacitated to promote any further signalling.…”

“…Although most of the previous efforts to explain the molecular function of DLC3-StART domain have failed (Alpy & Tomasetto, 2005), Beatriz Sanchez-Solana et al (Sanchez-Solana et al , 2021) proved that the DLC1-3 StART domains bind phosphatidylserine (PS). They also postulated that, in DLC1, the lipid-binding works as a mediator of the interaction with several proteins, independently from Rho-GAP domain activity (Sanchez-Solana et al ., 2021). In eukaryotic membranes, PS is a well-known phospholipid involved in signalling pathways (Kay & Grinstein, 2013).…”

“…proved that the DLC1-3 StART domains bind phosphatidylserine (PS). They also postulated that, in DLC1, the lipid-binding works as a mediator of the interaction with several proteins, independently from Rho-GAP domain activity (Sanchez-Solana et al, 2021). In eukaryotic membranes, PS is a well-known phospholipid involved in signalling pathways (Kay & Grinstein, 2013).…”

The START domain potentiates HD-ZIPIII transcriptional activity

“…One frequently used StARkin regulatory mechanism is modulation of homomeric stoichiometry 24,34,[39][40][41][42][43] .…”

DLC1 deficiency at diagnosis predicts poor prognosis in acute myeloid leukemia

“…DLC1 is a downregulated tumor suppressor gene and silenced by epigenetic mechanisms in human cancers, including several hematological malignancies [ 9 – 11 ]. Based on the bio-information database and external validation, our study suggests that DLC1 may be a potential marker affecting the prognosis of AML.…”