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The Conserved RING-H2 Finger of ROC1 Is Required for Ubiquitin Ligation

2000; Elsevier BV; Volume: 275; Issue: 20 Linguagem: Inglês

10.1074/jbc.m907300199

1083-351X

Angus Chen, Kenneth Wu, Serge Y. Fuchs, Peilin Tan, Carlos A. Gómez, Zhen‐Qiang Pan,

Glycosylation and Glycoproteins Research

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (ρ-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IκBα. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys 42 , Cys 45 , Cys 75 , His 77 , His 80 , Cys 83 , Cys 94 , and Asp 97 as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro . However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed.