Fig 2: UBR5 interacts with AKT and regulate its activity. a HEK293T cells were transiently transfected with FLAG-tagged UBR5 followed by IP by anti-FLAG antibody and Western Blot analysis. The full-length films are presented in Supplementary Fig. S5 [Panel 1–3]. b A549 cells were infected with the lenti-viruses from multiple shRNA construct against UBR5. The full-length films are presented in Supplementary Fig. S5 [Panel 4–5]. c Lung adenocarcinoma cell lines which are stably expressing Cas9 were transiently transfected with synthetic gRNAs against UBR5. The full-length films are presented in Supplementary Fig. S6

Fig 3: Cellular model of the proteostasis network for GABAAreceptors. Interactors that were identified from our SILAC analysis are included in each step. Interactors that are expected to play a role in the folding and assembly of GABAA receptors are colored in green; those in their degradation, in orange; and those in their transport, in purple. The GABAA receptor subunit is cotranslationally translocated to the ER membrane (state 1). The OST complex, including DDOST, RPN1, and RPN2, installs N-linked glycans to the subunit (state 1 to state 2). The glycoprotein is trimmed by ER α-glucosidases, such as GANAB (state 2 to state 3), and subjected to calnexin (CANX) folding cycles in a glycan-dependent manner (state 3 to state 4). In parallel, heat shock proteins and their cochaperones facilitate the folding of the subunit both in the ER lumen (HspA5, DNAJB11, and P4HB) and in the cytosol (HspA1A, HspA6, HspA8, DNAJA1, DNAJA2, and DNAJA3) (state 2 to state 4). After coordinated folding efforts (state 4), the subunit faces three possible routes. First, if folded into the native state, the subunit assembles with other subunits to form a heteropentamer with the assistance from assembly factors (state 4 to state 5). The assembled receptors engage the COPII machinery, including SEC16A, for anterograde trafficking to the Golgi and to the plasma membrane (state 5 to state 6 and state 7). The COPI machinery, including COPA, COPG2, aids the recycle of the subunits from the Golgi back to the ER (state 6 to state 5). Second, non-native subunits are reglucosylated by UGGT1 for additional folding attempts (state 4 to state 3). Third, terminally misfolded subunits undergo ERAD pathway, being recognized, ubiquitinated, dislocated, and targeted to the proteasome for degradation (state 4 to state 8 and state 9). Ubiquitin E3 ligases (UBR5, UBE3C, TRIM21, LTN1, and HUWE1) and other ERAD factors (Hsp90B1, Hsp90AB1, AHSA1, SEL1L, VCP, and ERLIN2) potentially play a critical role in this process. Aggregated subunits could utilize the lysosome-related degradation pathway (state 4 to state 10). ER, endoplasmic reticulum; ERAD, ER-associated degradation; GABAA, gamma-aminobutyric acid type A; OST, oligosaccharyltransferase; SILAC, stable isotope labeling by amino acids in cell culture; UGGT1, UDP-glucose glycoprotein glucosyltransferase 1.

Fig 4: WWP2 is a bona fide SOX2 E3 ubiquitin ligase in GSCs.A GSCs transduced with control or RNAi targeting CDH1, CUL4A, UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. 1I. Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. 3A. Data represent mean ± SEM (n = 3, unpaired t-test, **P < 0.01). See Source Data File for exact P values and statistical parameters.

Fig 5: UBR5 is altered in human lung adenocarcinoma. a UBR5 is altered by mutation or increased copy number in 10% of human lung adenocarcinoma analyzed by TCGA. b Schematic of UBR5 protein. UBR5 is a 2799 amino acid protein with an N-terminal UBA (ubiquitin-associated) domain, a middle zf-Box (zinc finger UBR-box) domain and two C-terminal domains, PABP (poly [A]-binding protein) & HECT (homology to E6-AP carboxyl terminus). c A wide variability in the UBR5 protein level was observed in various lung adenocarcinoma cell lines; Tubulin was used as a loading control. The full-length films are presented in Supplementary Fig. S2. d The level of UBR5 protein was found to be highly elevated in nearly all primary human lung tumor samples (T) compared to normal adjacent lung (N), N1 represents the normal adjacent to T1, N2 is the normal adjacent to T2, etc.; GAPDH was used as a house keeping gene. The full-length films are presented in Supplementary Fig. S3. e Densitometric analysis of western blot films from panel 1d as ratio of UBR5/GAPDH