Fig. 4

SIAH2 promotes the degradation of JAK2^V617F through the VxP motif in its kinase domain. a Upper panel, illustration depicting the organization of JAK2 into the structural JH1-7 domains and the distribution of VxP motifs within its four functional domains. Illustration was created with BioRender (https://BioRender.com). Lower panel, 3D visualization of JAK2 showing VxP motifs (yellow) and the ubiquitin-binding domain (orange) via ChimeraX V1.3. b SIAH2 was expressed as a glutathione S-transferase (GST) fusion protein, GST-SIAH2. Samples were purified via Glutathione Sepharose 4B beads and analyzed for JAK2 binding by immunoblotting. c Immunoprecipitates with anti-SIAH2 antibody or mouse preimmune serum (IgG) were analyzed for pJAK2 and JAK2 by immunoblotting. HEL cells were incubated with 50 nM bortezomib for 6 h to prevent the proteasomal degradation of SIAH2-bound JAK2^V617F. d VLP degron motif in the kinase domain determines JAK2 degradation by SIAH2. FLAG-tagged kinase domains of JAK2^WT or mutant JAK2^V1000G were coexpressed with GFP-SIAH2 in HEK293T cells. Immunoblotting verified the presence of the indicated proteins, and HSP90 served as a loading control. e HEL cells were transfected with siRNA against UBCH8 or noncoding siRNA. The cells were subsequently treated with 5 µM MERCK60 for 48 h. Immunoblotting was used to verify the expression of JAK2^V617F and UBCH8; GAPDH served as a loading control. f HEL cells were transfected as described in (e), stained with annexin-V-FITC/PI and analyzed for apoptosis via flow cytometry. g UBCH8 was expressed as the glutathione S-transferase (GST) fusion protein GST-UBCH8. The samples were purified via Glutathione Sepharose 4B beads and analyzed for JAK2 binding by immunoblotting. h Illustration summarizing how HDAC1 and HDAC2 protect JAK2^V617F from SIAH2-mediated proteasomal degradation. SIAH2 cooperates with UBCH8 and promotes polyubiquitylation and subsequent proteasomal degradation of JAK2^V617F. Illustration was created with BioRender (https://BioRender.com). The data are presented as mean ± SD of three independent experiments. Statistical analyses (one-way ANOVA; two-way ANOVA; ns not significant; Bonferroni correction; **P  < 0.01; ***P  < 0.001; ****P  < 0.0001)