Fig. 3: JAK2 interacts with BECN1 and phosphorylates BECN1 at Y333.

a, b Immunoprecipitation (IP) analyses were performed to examine the interaction between BECN1 and JAK2 (a) or JAK2 and BECN1 (b) in SW48 cells. c Co-IP was performed to examine the relationship between JAK2 and BECN1 in SW48 cells in the absence or presence of IL-6 (20 ng/ml) for 12 h (upper panels). Western blotting was performed on whole-cell extracts (WCEs) (lower panels). d Immunofluorescent staining (IF) analyses of LoVo cells using anti-BECN1 and anti-JAK2 antibodies. Yellow puncta, double-stained BECN1 and JAK2. Scale bars, 20 μm. e Co-IP was performed to examine the interaction of JAK2 and BECN1 in HEK293T cells cotransfected with Flag-BECN1 and HA-JAK2 (WT)/HA-JAK2 (K882E) (upper panels) for 24 h. Western blotting was performed on WCEs (lower panels). f Co-IP was performed to examine the tyrosine phosphorylation of Flag-BECN1 in HEK293T cells cotransfected with Flag-BECN1 and HA-JAK2 (WT)/HA-JAK2 (K882E) (upper panels) for 24 h. Western blotting was performed on WCEs (lower panels). g Co-IP analyses for HA-BECN1 and p-Tyr in HEK293T cells expressing Flag-JAK2, a vector control (Vector), HA-BECN1 WT, HA-BECN1 Y333F, or HA-BECN1 Y338F. Western blotting was performed on WCEs (lower panels). h Modeled complex structure between the ECD domain (magenta, left) and JH1 domain (right), where the JH1 domain consists of four regions: an N-terminal lobe (gray), a C-terminal lobe (cyan), a C helix (red), and an activation loop (yellow). ATP is colored by element, with carbon atoms in green, oxygen atoms in red, nitrogen atoms in blue, and phosphorus atoms in orange. The residues involved in the hydrophobic interaction are shown in pink sphere representation, with atoms shown as sticks. The hydrogen bond is shown by black dashed lines. On the bottom, from left to right, are the enlarged images for the interface in the catalytic site, the interface between the β sheet with the phosphorylation site of the ECD domain and the N-terminal lobe of the JH1 domain, and the interface between the hydrophobic loop of the ECD domain and the C-terminal lobe of the JH1 domain, respectively. i IP analyses for HA-BECN1 and p-BECN1 (Y333) in HEK293T cells expressing HA-BECN1 WT or HA-BECN1 Y333F. Western blotting was performed on WCEs (lower panels). j Western blotting was performed to examine the expression of BECN1 and p-BECN1 (Y333) in LoVo cells in the absence or presence of IL-6 (20 ng/ml) for 12 h. k SW48 cells were separately transfected with SiJAK2 and SiNC. After transfection for 48 h and following stimulation with IL-6 (20 ng/ml) for 12 h, western blotting was performed. Western blots are representative of two independent experiments. WCE whole-cell extract. Source data are provided in the Source Data file.