Figure 4: CK1-mediated phosphorylation of Beclin 1 is essential for Beclin 1 acetylation.

(a) Motif scan result of Beclin 1 by Scansite. Choosing the stringency level is high. (b) Cluster alignment of CK1 consensus phosphorylation motif on Beclin 1 proteins. Arrowheads point to T406 and S409 of human Beclin 1. (c) Mutations of T406 and S409 decrease Beclin 1 phosphorylation and acetylation. HEK293T cells were transfected with indicated Beclin 1 constructs. Beclin 1 phosphorylation, acetylation and the Beclin 1–Rubicon interaction were detected by IP as indicated. (d) Inhibition of CK1 with D4476 decreases Beclin 1 acetylation. HEK293T cells transfected with Flag-Beclin 1 were treated with indicated concentration of D4476 for 4 h. Beclin 1 acetylation and the interaction between Beclin 1 and p300 or Rubicon was determined with IP and western blot analyses. (e) CK1γ2 knockdown decreases the binding of p300 to Beclin 1 and Beclin 1 acetylation. Flag-Beclin 1 was co-transfected with siRNA into HEK293T cells. Beclin 1 acetylation and the interaction between Beclin 1 and p300 or Rubicon was determined with IP and western blot analyses. (f) Serum restoration increases Beclin 1 acetylation by activating CK1. HEK293T cells transfected with Flag-Beclin 1 were serum-starved for 24 h, and then serum was restored with or without D4476 pretreatment (40 μM, 2 h). (g) Overexpression of CK1γ2 increases WT Beclin 1 acetylation but not 2KR mutant. Flag-tagged Beclin 1 (WT, 2KR) was co-transfected with Myc-CK1γ2 or vector into HEK293T cells.