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, receptor interacting protein kinase 1 (RIP1) 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. Here, we used site-directed mutagenesis to systematically examine the effects of putative phosphor acceptor sites on RIP1 and RIP3 on TNF-induced programmed necrosis. We found that mutation of individual serine residues in the kinase domain of RIP1 had little effects on RIP1 kinase activity and TNF-induced programmed necrosis. Surprisingly, alanine substitution of Ser89 enhanced RIP1 kinase activity and TNF-induced programmed necrosis without affecting RIP1-RIP3 necrosome formation. This indicates that Ser89 is an inhibitory phospho-acceptor site that can dampen the pro-necrotic function of RIP1. In addition, we show that a phosphor-mimetic 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.

Source: BJ Signal - September 24, 2013 Category: Biochemistry Authors: T McQuade, Y Cho, F Ka-Ming Chan Tags: BJ Signal Source Type: research