Programmed necrosis or necroptosis is controlled by the action of two 
serine/threonine kinases, RIP1 (receptor-interacting serine/threonine protein 
kinase 1; also known as RIPK1) and RIP3. The phosphorylation of RIP1 and RIP3 is 
critical for assembly of the necrosome, an amyloid-like complex that initiates 
transmission of the pro-necrotic signal. In the present study, we used 
site-directed mutagenesis to systematically examine the effects of putative 
phosphoacceptor sites on RIP1 and RIP3 on TNF (tumour necrosis factor)-induced 
programmed necrosis. We found that mutation of individual serine residues in the 
kinase domain of RIP1 had little effect on RIP1 kinase activity and TNF-induced 
programmed necrosis. Surprisingly, an alanine residue substitution for Ser(89) 
enhanced RIP1 kinase activity and TNF-induced programmed necrosis without 
affecting RIP1-RIP3 necrosome formation. This indicates that Ser(89) is an 
inhibitory phosphoacceptor site that can dampen the pro-necrotic function of 
RIP1. In addition, we show that a phosphomimetic mutant of RIP3, S204D, led to 
programmed necrosis that was refractory to RIP1 siRNA and insensitive to 
necrostatin-1 inhibition. Our results show that programmed necrosis is regulated 
by positive and inhibitory phosphorylation events.