Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex 
(LUBAC) play an important role in NF-κB activation. However, the regulation of 
linear ubiquitin chain generation by LUBAC is not well characterized. Here, we 
identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear 
linkage specificity (OTULIN/Gumby/FAM105B) and cylindromatosis (CYLD) that can 
cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal 
PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP 
interacts with both CYLD and OTULIN even in unstimulated cells. The interaction 
of CYLD and OTULIN with HOIP synergistically suppresses LUBAC-mediated linear 
polyubiquitination and NF-κB activation. Moreover, introduction of a HOIP mutant 
unable to bind either deubiquitinase into HOIP-null cells augments the 
activation of NF-κB by TNF-α stimulation. Thus, the interactions between these 
two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling 
the extent of TNF-α-induced NF-κB activation in cells by fine-tuning the 
generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with 
OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling 
pathway activation by LUBAC. Our observations provide molecular insights into 
the roles of ligase-deubiquitinase interactions in regulating molecular events 
resulting from linear ubiquitin conjugation.