Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) -, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC- autophosphorylation, stimulation by TNF-, as well as cytokine stimulation of NF-B DNA binding and promoter activity. S¹⁴⁹ in hBVR S/T kinase domain and S²³⁰ in YLS²³⁰F in hBVR’s docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC-. Two hBVR-based peptides, KRNRYLS²³⁰F (#1) and KKRILHC²⁸¹ (#2), but not their SA or CA derivatives, respectively, blocked PKC- stimulation by TNF- and its membrane translocation. The C-terminal-based peptide KYCCSRK²⁹⁶ (#3), enhanced PKC- stimulation by TNF-; for this, Lys²⁹⁶ was essential. In metabolically ³²P-labeled HEK293 cells transfected with hBVR or PKC-, TNF- increased hBVR phosphorylation. TNF- did not stimulate PKC- in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide-binding loop (G¹⁷), S¹⁴⁹, or S²³⁰; this was similar to the response of "kinase-dead" PKC-^K281R. We suggest peptide #1 blocks PKC--docking site interaction, peptide #2 disrupts function of the PKC- C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC- activity and cellular response to cytokines.--Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E. M., Maines, M. D. Regulation of TNF--activated PKC- signaling by the human biliverdin reductase: Identification of activating and inhibitory domains of the reductase.