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. 2016 Jun 21;26(8):914–934. doi: 10.1038/cr.2016.78

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Copyright © 2016 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Plk3 phosphorylates procaspase-8 at T273. (A) Recombinant GST-His-fused Plk3 (0.1 μg) was incubated with recombinant active His-tagged caspase-8-p18 and -p10 (10 U) at 30 °C for the indicated time periods. The reaction mix was blotted against Plk3 (BD), GST and caspase-8. (B) Schematic representation of the recombinant GST-fused procaspase-8 (GST-Casp8-WT), N-terminal part (GST-NT), C-terminal subdomains of procaspase-8 (GST-p18 and -p10) and its mutated forms used for in vitro kinase assays. (C) Recombinant GST-fused procaspase-8 and its subdomains were incubated with Plk3 for an in vitro kinase assay and visualized by autoradiography. (D) HEK 293T cells were transfected with Flag-tagged Plk3-WT and its mutants (T219D, K91R). The proteins were immunoprecipitated using anti-Flag M2 affinity gel. Recombinant GST-fused caspase-8-p18 was incubated with immunoprecipitated Plk3 for an in vitro kinase assay, analyzed by SDS-PAGE and stained with Coomassie (lower panel), and visualized by autoradiography (upper panel). (E) GST-Casp8-WT, NT, p18 and point mutants of p18, all verified by sequencing in both directions, were incubated with recombinant GST-His-fused Plk3 (0.1 μg) for an in vitro kinase assay, analyzed by SDS-PAGE, stained with Coomassie (lower panel), and visualized by autoradiography (upper panel). (F) Recombinant GST-fused p18-WT, p18 T273A and increasing amounts of Casp8-WT and Casp8 T273A were incubated with recombinant GST-His-fused Plk3 (0.1 μg) for an in vitro kinase assay, analyzed by SDS-PAGE, stained with Coomassie (lower panel), and visualized by autoradiography (upper panel).