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. Author manuscript; available in PMC: 2014 Sep 15.
Published in final edited form as: Biochem J. 2013 Dec 15;456(3):409–415. doi: 10.1042/BJ20130860

Figure 1. The effect of serine phosphorylation on RIP1 kinase activity.

Figure 1

(A) Effect of alanine substitutions of reported phosphorylation sites on RIP1 kinase activity. HEK293T cells were transfected with the indicated GFP-tagged RIP1 plasmids. The in vitro kinase activity was determined using RIP1 autophosphorylation as readout. The numbers in parentheses represent kinase activity normalized to expression level of RIP1-GFP on bottom panel. (B) Ser161 does not control the pro-necrotic function of RIP1. RIP1-deficient Jurkat cells were transfected with the indicated RIP1-GFP plasmids or empty GFP vector. Transfected GFP+ cells were tested for TNF-induced apoptosis or TNF and zVAD-induced programmed necrosis. (C) Ser161 controls sensitivity to Nec-1 inhibition. RIP1-deficient Jurkat cells stably expressing wild type RIP1-GFP or S161A-RIP1-GFP were treated with TNF, zVAD, and the indicated doses of Nec-1. (D) RIP1-deficient Jurkat cells were transfected with the indicated RIP1-GFP plasmids. Transfected GFP+ cells were tested for TNF and zVAD-induced programmed necrosis. p values were calculated by comparing WT transfectants with the indicated mutants using Student’s t test.