Fig. 2 | Nature Communications

Fig. 2

From: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

Fig. 2The alternative text for this image may have been generated using AI.

CaMKII directly phosphorylates Beclin 1 at Ser90. a The phosphorylation of Beclin 1-S90 requires activated CaMKII. SK-N-SH cells were transiently transfected with a control vector or plasmids encoding His-CaMKII-KD for 48 h. The whole-cell extracts were subjected to western blotting using the indicated antibodies. b CaMKII is required for phosphorylation of Beclin 1 on S90. HEK293T cells were transiently transfected with negative control or CaMKII shRNA, and then untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 72 h. The cells were lysed and the indicated proteins were analyzed by western blot. c Increased association between CaMKII and Beclin 1 in cells treated with ionomycin or EB1089. HEK293T cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, lysed, and then incubated with anti-CaMKII or control IgG antibodies prior to western blotting. The resulting immunoprecipitates (IP) were subjected to western blot analysis. TCL, the whole-cell lysate. d The association between His-tagged CaMKII and Flag-tagged Beclin 1. HEK293T cells or SK-N-SH cells were transiently co-transfected with plasmids encoding Flag-Beclin 1-WT and His-CaMKII-WT, lysed, and incubated with an anti-Flag antibody prior to western blot. The resulting immunoprecipitates were subjected to western blot analysis. TCL, the whole-cell lysate. e Phosphorylation of Beclin 1 by CaMKII upon ionomycin or EB1089 treatment. HEK293T cells were transiently transfected with plasmids encoding Flag-Beclin 1-WT, and the cells were incubated in 6 μM ionomycin or 100 nM EB1089. Flag-tagged proteins were precipitated and analyzed by western blot using an anti-phospho-S90-Beclin 1 and other indicated antibodies. f The phosphorylation-dependent activity of CaMKII on Beclin 1. HEK293T cells transfected with His-CaMKII-WT were untreated or treated with 6 μM ionomycin or 10 μM KN-93 for 24 h, then lysed and incubated with an anti-His antibody. Purified Beclin 1 fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed. g In vitro phosphorylation of Beclin 1. HEK293T cells transfected with His-CaMKII-WT were treated with 6 μM ionomycin, then lysed and incubated with an anti-His antibody. Purified Beclin 1-WT and Beclin 1-S90A fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed

Back to article page