In resting cells, IkB proteins are responsible for sequestering NFkB subunits in the cytoplasm. Phosphorylation by the IKK complex targets IkB proteins for ubiquitination and degradation, allowing NF-kB to enter the nucleus and activate gene expression. CD40-mediated phosphorylation and degradation of IkBa in HOIP-deficient cells was dramatically impaired relative to that observed in parental A20.2J cells. We also assayed activation of the stress-activated protein kinase JNK in response to CD40 engagement. CD40-mediated JNK activation in HOIP-deficient cells was impaired as measured by phosphorylation of Thr183 and Tyr185 in JNK. CD40-induced activation of NF-kB and JNK in HOIP-reconstituted cells was normal, demonstrating that the defects observed in gene-deficient cells were due to the absence of HOIP expression. The marked defects in CD40-mediated cell activation and WY 14643 signaling displayed by HOIP-deficient cells suggested that HOIP mediates recruitment of critical components of the CD40 signaling apparatus to the receptor. Previously, we demonstrated that HOIP is recruited to the CD40 signaling complex in a TRAF2- dependent manner, suggesting that HOIP functions downstream of TRAF2. Studies by others suggest that the TRAF2- associated proteins cIAP1 and cIAP2 play a role in the recruitment of HOIP to TNFR1 and CD40. Therefore, we determined whether the association of HOIP with CD40 in A20.2J cells was altered by treatment of cells with an inhibitor of cIAP activity, a membrane-permeable peptide derived from the apoptosis regulator SMAC. We found that pretreatment of cells with the SMAC peptide dramatically reduced the amount of cIAP1 associated with the CD40 signaling complex in cells stimulated with anti-CD40 antibody-coated beads. SMAC peptide treatment also resulted in a slight but reproducible decrease in the amount of the major HOIP form recovered by CD40 immunoprecipitation, along with an apparent increase in higher molecular weight species recognized by anti-HOIP antibody. In contrast, treatment with the SMAC peptide did not alter the amount or molecular weight of HOIP present in cell lysates. These data suggest that SMAC peptide treatment specifically alters the characteristics of CD40-associated HOIP rather than the entire cellular pool of this protein. Together, these results support the idea that the cIAP proteins influence the recruitment and posttranslational modification state of CD40-associated HOIP. To test the possibility that HOIP is responsible for the recruitment of other critical signaling proteins to CD40.