The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation.
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 (rho-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 IkappaBalpha. 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.
Chen
A
A
Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Wu
K
K
Fuchs
S Y
SY
Tan
P
P
Gomez
C
C
Pan
Z Q
ZQ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine; D006801:Humans; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008627:Mercuric Chloride / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D006902:Hydroxymercuribenzoates; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D014452:Ubiquitins; C030601:4-hydroxymercuribenzoate; D008627:Mercuric Chloride; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D003545:Cysteine