Fig 2: Identification of ubiquitin-modified lysine residues on EGFR mediated by ZNRF1 and CBL in response to EGF. (A) Schematic diagram of EGFR TKD sequence and its 15 lysine residues. (B–E) HeLa cells were co-transfected with wild type or various EGFR mutants in combination with an empty vector or the E3 ubiquitin ligase ZNRF1 or CBL as indicated for 48 h. Cells were serum-starved and stimulated with 100 ng/mL EGF for 5 min. Ubiquitination of EGFR was examined by immunoprecipitation with Myc-conjugated agarose, followed by immunoblotting with anti-ubiquitin and anti-Myc antibodies. The experiments in (B,C) were repeated two times with similar results. Quantification of immunoblotting analysis data of three independent experiments are shown in the left panels of (D,E). Results are presented as averages ± SEM. n.s., no significant; *P < 0.05, **P < 0.01 (Student’s t-test).

Fig 3: Cbl directly ubiquitinates JAK2 via the K63 linkage. (a) Cbl is involved in cytokine-triggered K63-linked ubiquitination of JAK2. After overnight serum starvation, parental TF1 cells (TF1) or Cbl knockout TF1 cells (CKO2 and CKO17) were processed and analysed as described in Fig. 1b. (b) Ectopic expression of Cbl enhances JAK2 ubiquitination. JAK2 and Flag-tagged ubiquitin were co-expressed in 293 T cells with or without HA-Cbl as described previously. WCL were prepared and analysed as described in Fig. 1c. A short and longer exposure of the JAK2 Western blot is shown for clarity. (c) Cbl rescues high molecular weight signals of JAK2 ubiquitination. JAK2 and Flag-tagged ubiquitin were co-expressed in Cbl knockout GMR HeLa (CKO) cells with or without HA-Cbl (wide type or C381A, a ligase-deficient mutant), then subjected to analysis as described in Fig. 1c. (d) JAK2 is directly ubiquitinated by Cbl in an in vitro assay. For the in vitro ubiquitination assay, Flag-tagged JAK2 and HA-tagged WT or C381A mutant Cbl were prepared as described in the Methods, and these were then incubated with recombinant E1, E2 (Ubc13), or both together, in the presence of biotin-labelled ubiquitin. After incubation, reactions were directly analysed by western blot with the indicated antibodies.

Fig 4: Gab1, Grb2, Shc, and Shp2 adaptors mediate candidalysin-induced signaling events that trigger oral epithelial cytokine responses. C. albicans infections are initiated by increased fungal burden, associated hypha formation, and secretion of the pore-forming toxin candidalysin (Clys). When accumulated at high concentrations, Clys interacts with an epithelial cell membrane to form pore-like structures that result in membrane damage, alarmin release, and calcium influx. These events lead to activation of matrix metalloproteinases (MMPs) and the cleavage/release of epidermal growth factor receptor (EGFR) ligands. EGFR activation leads to the recruitment and activation of the Gab1, Grb2, Shc, Shp2, and c-Cbl adaptor proteins which in turn mediate MKP1/DUSP1 and c-Fos activation via the MAPK-ERK1/2 signaling pathway. Cumulatively, this leads to induction of GM-CSF and G-CSF, as well as IL-6, IL-1a, and IL-1ß, which mediate the subsequent stages of the innate and adaptive immune responses. Figure created with Biorender.com

Fig 5: Cbl plays a positive role in GMRß/JAK2/STAT5 signal axis in cells. (a,b) Cbl affects cytokine-induced JAK2 and STAT5 activation in GMRaß HeLa (GMR) cells. (a) HA-tagged Cbl (wide type or C381A, a ligase-deficient mutant) were transiently expressed in Cbl knockout GMR cells (CKO). After serum starvation for 16 h, transfected cells, as well as the parental GMR cells, were stimulated with (+) or without (-) GM-CSF (GM) (1 ng/mL) for 10 min, and their WCL were analysed by Western blot using the indicated antibodies. (b) Quantification of the data from (a). (c) Cbl promotes STAT5-driven CISH reporter activity upon GM-CSF stimulation. CKO and parental GMR cells were transiently co-transfected with the indicated wild-type (WT) or mutant (mut) CISH reporter genes and STAT5a without (lanes 5–12) or with (lanes 13–20) Cbl rescue. Transfected cells were treated with (+), or without (-), GM-CSF (GM) (1 ng/mL) for 24 h prior to performing the luciferase assay. The lower panel illustrates the protein levels of endogenous and overexpressed STAT5 and Cbl, as well as tubulin for normalization, in the various transfected cells. (d) The ubiquitin ligase activity of Cbl is required for rescuing STAT-dependent reporter activity. Cells were transfected and analysed as described in (c), except that cells were rescued with WT Cbl (lanes 5–8) or a ubiquitin E3 ligase dead mutant (C381A Cbl) (lanes 9–12). In (c) and (d), the relative luciferase values in the upper panel are presented as mean ± SEM. All experiments were repeated three times. Statistical significance (*P < .05, **P < 0.01 and ***P < 0.001) was determined by t-test.