Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination

Abstract

The leucine zipper-like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

Fig. 1.. LZTR1 loss recapitulates disease phenotypes.

(A) Morphometric characteristics of the skulls of 12-month-old Lztr1^+/+ and Lztr1^+/− male mice. (B) Haematoxylin and eosin–stained heart ventricular sections. Scale bar, 0.5 mm. The total cardiac area was quantified by Fiji. In the graph, horizontal lines represent means ± SD. (C) A mean area of 200 cardiomyocytes measured in laminin-stained heart sections of Lztr1^+/+ and Lztr1^+/− mice. Horizontal lines represent means ± SD. (D) Growth rate of early-passage MEFs isolated from three Lztr1^+/+ and three Lztr1^−/− embryos. (E) Growth rate of Lztr1^−/− MEFs expressing an empty vector (EV), wt-LZTR1, or LZTR1 mutants. n = 3. (F) AI growth of Schwann cells expressing Cas9 or Cas9/gLZTR1 (gLZTR1, guide RNA targeting LZTR1). n = 3. (G) AI growth of LZTR1-indel Schwann cells expressing the indicated constructs. n = 3. M202R, Met²⁰²→Arg. (H) Quantitative real time polymerase chain reaction (qRT-PCR) analysis of mRNA expression in primary human Schwann cells expressing shGFP or pooled shLZTR1. n = 3. For (D) to (H), values are means ± SEM. For (A) to (C) and (F) to (H), P values are from a two-sided Student’s t test. For (D) and (E), P values were detected by two-way analysis of variance (ANOVA).

Fig. 2.. The LZTR1-CUL3 complex ubiquitinates RAS proteins.

(A and B) Virotrap screens performed in HEK293T cells using group-specific antigen (GAG)–LZTR1 (A) or GAG-HRAS–deltaCAAX (deltaC) (B) as baits. Escherichia coli dihydrofolate reductase (eDHFR) fused to GAG was used as a negative control. (C) A scheme for the generation of cells expressing in-frame HiBiT-LZTR1 protein. RAS was immunoprecipitated from the HiBiT-LZTR1 edited HeLa cell lysates with panRAS antibody. Luminescent signal was generated by HiBiT incubated with LgBiT. nt, nucleotide. (D) Ubiquitinated RAS was purified from HEK293T cells expressing the indicated constructs by Co²⁺ metal affinity chromatography and detected by immunoblotting. Numbers on the left are molecular masses in kDa. 2xUb, two Ub; 1xUb, one Ub; EV, empty vector, WCL, whole-cell lysate; ns, nonspecific. (E) A workflow for the ubiquitome analysis. LC-MS/MS, liquid chromatography–tandem mass spectrometry. (F) A heatmap showing differentially ubiquitinated peptides in Lztr1^+/+ and Lztr1^−/− MEFs. The scale shows Z-scored site intensity values. (G) The quantification of the PLA analysis of Schwann cells expressing Cas9 or Cas9/gLZTR1 using antibodies against panRAS and Ub. Values are means ± SEM; n = 3. P values are from a two-sided Student’s t test. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3.. Disease-associated LZTR1 mutations are loss of function.

(A) LZTR1 mutations in schwannomatosis and Noonan syndrome individuals. Missense LZTR1 mutations identified in our cohort of schwannomatosis patients are shown in blue. In the schematic, K indicates Kelch domain. See Fig. 2 legend for amino acid abbreviations. (B) Flag-tagged CUL3 purified from HEK293T cells was incubated with HA-tagged LZTR1-overexpressing cell lysates and then immunoprecipitated using anti-Flag resin. LZTR1 was detected by immunoblotting with anti-HA antibody. (C) Cross-linking reactions were performed using HA-tagged LZTR1 purified from HEK293T cells. LZTR1 was detected by immunoblotting using anti-HA antibody. (D) Immunostaining of HeLa cells expressing HA-tagged wt-LZTR1 or LZTR1 mutants with anti-HA antibody. Scale bar, 10 μm. (E) RAS proteins were immunoprecipitated with antibody against panRAS. LZTR1 was detected by immunoblotting with anti-HA antibody. (F) Colocalization of mCherry-NRAS and HA-tagged LZTR1 expressed in HeLa-Cas9/gLZTR1 cells. Values are means ± SEM. P values were detected by two-sided Student’s t test. (G) Ubiquitinated NRAS was purified from HEK293T cells expressing the indicated constructs by Co²⁺ metal affinity chromatography and detected by anti-Flag antibody.

Fig. 4.. Ubiquitination at K170 inhibits RAS by impairing its association to the membrane.

(A) MEFs isolated from three Lztr1^+/+ and three Lztr1^−/− embryos were serum-starved, stimulated with 10% serum, and analyzed by immunoblotting. Values are means of phosphorylated (p) relative to nonphosphorylated protein levels ± SEM. P values are from a two-way ANOVA. (B) Progeny from the indicated Lztr1^+/− matings. Pregnant mice were treated with pimasertib starting from E7.5. (C) Quantitative ubiquitome analysis of Lztr1^+/+ and Lztr1^−/− MEFs. FDR, false discovery rate. (D) Ubiquitinated RAS was purified from HEK293T cells expressing the indicated constructs by Co²⁺ metal affinity chromatography and detected by immunoblotting. (E) The PLA analysis of wt-Hras and Hras-K170R MEFs expressing shGFP or shLztr1 using antibodies against panRAS and Ub. Red, PLA signal; blue, 4′,6-diamidino-2-phenylindole; scale bar, 10 μm. (F) HEK293T cells expressing the indicated constructs were serum-starved overnight, stimulated with 10% serum, and analyzed by immunoblotting. Values are means of phosphorylated relative to nonphosphorylated protein levels ± SEM; n = 3. P values are from a two-way ANOVA. (G) Snapshots of Ub-conjugated RAS at the lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids (3:1 molar ratio). (H and I) Immunoblotting of the membrane and cytoplasmic fractions isolated from HeLa cells expressing shGFP and shLZTR1 (H) or wt-Hras and Hras-K170R MEFs (I). HSP70, heat shock protein 70.