PhosphoPlus RIP (Ser166) Antibody Duet

The receptor-interacting protein (RIP) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that trigger pro-survival and inflammatory responses through the activation of NF-κB, as well as pro-apoptotic pathways (1). In addition to the kinase domain, RIP contains a death domain responsible for interaction with the death domain receptor Fas and recruitment to TNF-R1 through interaction with TRADD (2,3). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (4,5). RIP also interacts with TNF-receptor-associated factors (TRAFs) and can recruit IKKs to the TNF-R1 signaling complex via interaction with NEMO, leading to IκB phosphorylation and degradation (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (2,3). Caspase-8-dependent cleavage of the RIP death domain can trigger the apoptotic activity of RIP (8).Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury (9,10). The process is negatively regulated by caspases and is initiated through a complex containing the RIP and RIP3 kinases, typically referred to as the necrosome. Necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (11). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury (12). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (13). 1.Meylan, E. and Tschopp, J. (2005) Trends Biochem Sci 30, 151-9. 2.Hsu, H. et al. (1996) Immunity 4, 387-96. 3.Stanger, B.Z. et al. (1995) Cell 81, 513-23. 4.Ting, A.T. et al. (1996) EMBO J 15, 6189-96. 5.Kelliher, M.A. et al. (1998) Immunity 8, 297-303. 6.Devin, A. et al. (2000) Immunity 12, 419-29. 7.Zhang, S.Q. et al. (2000) Immunity 12, 301-11. 8.Lin, Y. et al. (1999) Genes Dev 13, 2514-26. 9.Christofferson, D.E. and Yuan, J. (2010) Curr Opin Cell Biol 22, 263-8. 10.Kaczmarek, A. et al. (2013) Immunity 38, 209-23. 11.Degterev, A. et al. (2008) Nat Chem Biol 4, 313-21. 12.Degterev, A. et al. (2005) Nat Chem Biol 1, 112-9. 13.Ofengeim, D. and Yuan, J. (2013) Nat Rev Mol Cell Biol 14, 727-36.