Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis.
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.
*Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
Thomas
T
McQuade
Youngsik
Y
Cho
Francis Ka-Ming
FK
Chan
D019943:Amino Acid Substitution; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D019169:Jurkat Cells; D020125:Mutation, Missense; D009336:Necrosis; D028861:Nuclear Pore Complex Proteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D016601:RNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
C119724:AGFG1 protein, human; D007093:Imidazoles; D007211:Indoles; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; C507699:necrostatin-1; C119705:RIPK3 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases