Fig. 2: JOSD2 deubiquitinates and stabilizes KRAS in an enzyme activity-dependent manner. | Nature Communications

Fig. 2: JOSD2 deubiquitinates and stabilizes KRAS in an enzyme activity-dependent manner.

From: Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer

Fig. 2

a The ability of KRASG12C/G12D/G12V to bind to JOSD2 was stronger than that of KRASWT. WT, wild-type; IP, immunoprecipitation. b The interaction between JOSD2 and KRAS was independent of the enzyme activity of JOSD2. CA&HY, JOSD2-C24A&H125Y. c–e JOSD2, not JOSD1, interacted with KRAS at semi-endogenous levels and endogenous levels. f In vitro GST pull-down assays to examine the interaction between JOSD2 and KRASWT/G12C/G12D/G12V. 293T cells were transfected with the indicated expression plasmids for 24 h. Cell lysates were incubated with the Streptavidin magnetic beads conjugated with bacterial-expressed recombinant GST or GST-JOSD2 protein. Proteins retained on Streptavidin magnetic beads were subjected to WB analysis. g, h JOSD2, not JOSD1, removed the polyubiquitination chains of KRASWT/G12C/G12D/G12V in an enzyme activity-dependent manner. i JOSD2 depletion promoted the polyubiquitination of KRASWT/G12C/G12D/G12V. j JOSD2 removed the polyubiquitination chains of KRASWT/G12C/G12D/G12V in vitro (The schematic diagram was created in BioRender. https://BioRender.com/w35e177). 293T cells were co-transfected with indicated expression plasmids, and cell lysates were incubated with anti-Flag affinity gel overnight. Then, proteins retained on anti-Flag affinity gel were incubated with bacterial-expressed recombinant human JOSD2 (rhJOSD2) protein at 37 °C for 3 h, followed by WB analysis. k JOSD2, not JOSD1, removed the polyubiquitination chains of KRASG12V promoted by LZTR1. The samples derived from the same experiment and processed on different gels in parallel were shown below: d Input: one gel for JOSD1/JOSD2/GAPDH, another for Flag(G12V), IP: one gel for JOSD1/Flag(G12V), another for JOSD2; e SW620: Input: one gel for JOSD1/KRAS/GAPDH, another for JOSD2, IP: one gel for JOSD1/KRAS, another for JOSD2; HCT-116: Input: one gel for JOSD2/GAPDH, another for JOSD1/KRAS, IP: different gels for JOSD1, KRAS and JOSD2; f one gel for KRAS/GST-JOSD2, another for GST; g Input of KRAS-WT/G12C: one gel for HA(ub)/GAPDH/ Flag(KRAS), another for JOSD1&2, Input of KRAS-G12D/G12V: one gel for HA(ub)/GAPDH/JOSD1&2, another for Flag(KRAS); i Input of KRAS-WT/G12C/G12D/G12V: one gel for HA(ub)/GAPDH/JOSD2, another for Flag(KRAS); k Input: one gel for HA(ub)/GAPDH, another for Flag(KRAS), another for Myc(LZTR1)/JOSD1&JOSD2. Source data are provided as a Source Data file.

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