Figure 4: CKIδ phosphorylates Set8 at S253 site.
From: SCFβ-TRCP promotes cell growth by targeting PR-Set7/Set8 for degradation
(a) Alignment of the candidate phosphodegron sequence in Set8 from different species. (b) Immunoblot (IB) analysis of cells transfected with Flag-β-TRCP1 and HA-tagged wild type or the S253A mutant Set8 constructs, as indicated. Where indicated, cells were treated with Myc-CKIδ, or the proteasome inhibitor MG132. (c) IB analysis of WCL and IP derived from HeLa cells transfected with Flag-β-TRCP1, HA-WT-Set8 and HA-S253A-Set8. (d) A schematic illustration of Set8 functional domains and the truncation mutants with different domains (C, M, N domain) that are used in e. (e) IB analysis of WCL and IP derived from HeLa cells transfected with Flag-β-TRCP1, HA-WT-Set8 and HA-Set8 constructs with different domains (C, M, N domain). (f) IB analysis of HeLa cells transfected with Flag-β-TRCP1, Myc-CKIδ and Flag-tagged wild-type or ΔPIP mutant Set8 constructs, as indicated. (g) IB analysis of HeLa cells transfected with HA-β-TRCP1, Myc-CKIδ and Flag-tagged ΔPIP mutant Set8 constructs. Where indicated, 10 μM MG132 was added for 12 h before collecting. (h) IB analysis of WCL and IP derived from HeLa cells transfected with HA-β-TRCP1 and indicated Flag-Set8 constructs. (i) IB analysis of WCL and IP derived from 293 T cells transfected with Flag-Set8 mutants and Myc-tagged Cullin1. (j) IB analysis of WCL and IP derived from 293 T cells transfected with Flag-Set8 and HA-β-TRCP1 and treated with D4476 under UVC exposure condition. (k) In vivo ubiquitination assays to demonstrate that SCFβ-TRCP promotes Set8 ubiquitination in a pSer253-dependent manner. His-pull-downed ubiquitinated Set8 and WCL were subjected to IB analysis with indicated antibodies.
