Brooke-Spiegler syndrome, familial cylindromatosis, and familial 
trichoepithelioma are autosomal-dominant genetic predispositions for benign 
tumors of skin appendages caused by mutations in the CYLD gene localized on 
chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific 
protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase 
(JNK) signaling. We investigated five families affected with these skin 
neoplasms and identified four premature stop codons and the novel missense 
mutation D681G in a family in which 11 of 12 investigated tumors were 
trichoepitheliomas. CYLD protein harboring this missense mutation had a 
significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- 
and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha 
(TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was 
coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin 
chains. Aspartic acid 681 is highly conserved in CYLD homologues and other 
members of the UBP family, but does not belong to the Cys and His boxes 
providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported 
previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond 
with the C-terminal end of ubiquitin and is important for the enzymatic 
activity. These results underline that D681 in CYLD is required for cleavage of 
K63-linked polyubiquitin chains.