The linear ubiquitin chain assembly complex (LUBAC) is well known as a major upstream activator of the transcription factor NF-kappaB in response to inflammatory and anti-apoptotic signals. It is composed of the active E3 ligase HOIP (HOIL-1L interacting protein) and two other subunits SHARPIN (shank-associated RH domain-interacting protein) and HOIL-1L (heme-oxidized iron regulators protein 2 ubiquitin ligase-1). Thus far, little is known concerning the regulation of HOIP catalytic activity. Our results suppose that mechanstically HOIP functions as a RING/HECT-hybrid as it was recently proposed for several RBR-type E3s. In substitution experiments, we could show that HOIL-1L RBR cannot compensate for HOIP RBR resulting in a catalytically inactive HOIP chimera. A yeast two-hybrid screen using a human E2 library identified distinct yet overlapping interactors for HOIP and HOIL-1L. By mutational analyses of particular lysine residues we currently investigate the potential effect of HOIP auto-ubiquitination on NF-kappaB activation. Taken together, our data suggests an intricate mechanism of LUBAC regulation.