ROC1, a Homolog of APC11, Represents a Family of Cullin Partners with an Associated Ubiquitin Ligase Activity

Abstract

We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

“…In budding yeast, it is known that there is only one member of the ySAG/yROC/yRbx/Hrt family. Targeted disruption of this gene results in cell death that can be fully rescued by ROC1/Rbx1 Ohta et al, 1999) or ySAG (this report) or ROC2, an N-terminal truncated SAG, as reported (Ohta et al, 1999). Since both ROC1/Rbx1/Hrt1 and SAG has ubiquitin ligase activity when complexed with SCF component, it is likely that SAG/ROC/Rbx/Hrt regulates cell cycle through ubiquitin/proteolysis pathway.…”

“…We showed a morphological cell enlargement and a broad distribution of¯uorescence intensities with an increased DNA content in SAG knockout cells (Figure 4). Several recent ®ndings also support this notion: (1) Xenopus Cdc34, an E2 that binds to SAG/ROC/Rbx/Hrt Ohta et al, 1999;Seol et al, 1999;Skowyra et al, 1999;Tan et al, 1999;Tyers and Willems, 1999) promoted degradation of Wee1 (Michael and Newport, 1998), a key G2/M negative regulator (McGowan and Russell, 1993;Parker and Piwnica Worms, 1992); (2) SCF components, Skp1 and Cul1 were localized to the centrosome and regulated the centrosome duplication cycle in mammalian cells (Freed et al, 1999); and (3) Cdc4, an F-box protein in SCF complex was required for the onset of S phase as well as anaphase in Saccharomyces cerevisiae (Goh and Surana, 1999).…”

“…Among genes that are involved in G1?S progression, Cul1 is a SAG/ROC binding protein and a component of E3 SCF complex required for SIC-1 degradation Ohta et al, 1999;Willems et al, 1996). CLG1 is a cyclin-like protein that interacts with Pho85p (Matsumoto and Wickner, 1993), a Ser/Thr kinase involved in induction of G1?S progression (Espinoza et al, 1994;Levine et al, 1995).…”

“…SAG is also shown to be a component of E3 ubiquitin ligase involved in the degradation of many biological important molecules including IkB Ohta et al, 1999;Skowyra et al, 1999;Tan et al, 1999). It is possible that anti-apopotosis activity of SAG/ROC/Rbx can also be achieved through ubiquitination and degradation of IkB, an inhibitor of NF-kB (Ohta et al, 1999;Tan et al, 1999), leading to activation of apoptosis inhibitor, NF-kB (Beg and Baltimore, 1996;Mayo et al, 1997;Van Antwerp et al, 1996, 1998Wang et al, 1996). This hypothesis is being tested currently in our laboratory.…”

“…While this manuscript was in its late stage of preparation, several groups have reported the cloning and characterization of ROC1 (regulator of cullins), Rbx1 (RING box) or Hrt1 that either binds to cullins or VHL tumor suppressor gene product or SCF complex to form important E3 ubiquitin ligase complex Ohta et al, 1999;Seol et al, 1999;Skowyra et al, 1999;Tan et al, 1999;Tyers and Willems, 1999). By direct sequencing comparison, SAG turns out to be the second member of this ROC/Rbx/Hrt family (ROC2/Rbx2/Hrt2).…”