Selective regulation by δ-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils

TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Antiapoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when beta-integrin signaling is activated, TNF phosphorylates TNFR-1 and activates prosurvival and antiapoptotic signaling. Previously, we identified the delta-PKC isotype and phosphatidylinositol (PI) 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NF-kappaB activation, and inhibition of apoptosis. The purpose of this study was to examine the role of delta-PKC and PI 3-kinase in the assembly of TNFR-1 signaling complex that regulates NF-kappaB activation and antiapoptotic signaling. Coimmunoprecipitation studies established that PI 3-kinase, delta-PKC, and TNFR-1 formed a signal complex in response to TNF. delta-PKC recruitment required both delta-PKC and PI 3-kinase activity, whereas PI 3-kinase recruitment was delta-PKC independent, suggesting that PI 3-kinase acts upstream of delta-PKC. An important regulatory step in control of antiapoptotic signaling is the assembly of the TNFR-1-TNFR-1-associated death domain protein (TRADD)-TNFR-associated factor 2 (TRAF2)-receptor interacting protein (RIP) complex that controls NF-kappaB activation. Inhibition of either delta-PKC or PI 3-kinase decreased TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus delta-PKC and PI 3-kinase are positive regulators of TNF-mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that delta-PKC and PI 3-kinase regulate TNF antiapoptotic signaling at the level of the TNFR-1 through control of assembly of a TNFR-1-TRADD-RIP-TRAF2 complex.