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 Ser89 enhanced RIP1 kinase activity and TNF-induced programmed necrosis without affecting RIP1–RIP3 necrosome formation. This indicates that Ser89 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.
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Research Article|
November 22 2013
Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis
Thomas McQuade;
Thomas McQuade
1
*Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
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YoungSik Cho;
*Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
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Francis Ka-Ming Chan
Francis Ka-Ming Chan
3
*Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
†Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
3To whom correspondence should be addressed (email Francis.chan@umassmed.edu).
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Publisher: Portland Press Ltd
Received:
June 28 2013
Revision Received:
September 19 2013
Accepted:
September 24 2013
Accepted Manuscript online:
September 24 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 456 (3): 409–415.
Article history
Received:
June 28 2013
Revision Received:
September 19 2013
Accepted:
September 24 2013
Accepted Manuscript online:
September 24 2013
Citation
Thomas McQuade, YoungSik Cho, Francis Ka-Ming Chan; Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis. Biochem J 15 December 2013; 456 (3): 409–415. doi: https://doi.org/10.1042/BJ20130860
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