A Novel RING Finger Protein, Human Enhancer of Invasion 10, Alters Mitotic Progression through Regulation of Cyclin B Levels

Abstract

The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle. The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation. Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins. Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G 2 /M transition. In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts. Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast. HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site. Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro. These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G 2 /M.The spatial and temporal regulation of cell division is essential for development and normal cellular replacement in both unicellular and multicellular organisms. Cell cycle control and cell morphology are coordinated by complex signaling networks, the inappropriate perturbation of which can lead to either programmed cell death (apoptosis) or indefinite cell division cycles (cancer). The fact that many protein constituents of such pathways are conserved over great phylogenetic distances among eukaryotes emphasizes their importance. Since the 1980s, complementation studies in which human genes were identified based on the specific rescue of, or genetic interaction with, orthologous pathways in yeast have proven to be a fruitful means of gaining insight into mammalian growth controls. For example, the central cell cycle kinases CDC2 (32) and CDK2 (11) and the C, D, and E cyclins (15,24,53,57) were identified by complementation of yeast mutants.Analysis of yeast budding controls has been of particular interest as a model of cellular morphogenesis and has provided an interpretive framework for studies of signal transduction and morphological control processes in less experimentally amenable multicellular organisms (see, for example, reference 26). Bud emergence in vegetatively growing yeast is a complex process that depends on the rearrangement of the cytoskeleton throughout the different phases of the cell cycle (8, 56). The site of bud emergence is specified by signals that mark the location of a previous bud. In addition to the spatial regulation of budding, bud emergence is temporally synchronized with the different phases of the cell cycle and is sensitive to the environment. A coordinated response to change...

Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor

“…High levels of Cyclin B1 might cause misregulation of the Cyclin B1/Cdk1 complex, allowing escape from the G2-M checkpoint induced by DNA damage (Lobrich and Jeggo, 2007). Interestingly, and compatible with this possibility, CCNB1IP1 is needed to maintain the accurate euploid segregation at the meiotic prophase I-metaphase I boundary (Ward et al, 2007) and the mitotic G2-metaphase boundary (Toby et al, 2003).…”

“…Some of the known molecular functions of CCNB1IP1 would support a tumor suppressor role. This ligase controls the degradation of Cyclin B1 (Toby et al, 2003), projecting elevated levels of this cyclin in CCNB1IP1-deficient cells. Overexpression of Cyclin B1, in turn, has been reported in various human tumors, and it is a predictor of poor prognosis (Wang et al, 1997;Soria et al, 2000;Dong et al, 2002;Hassan et al, 2002;Yasuda et al, 2002).…”

HEI10 negatively regulates cell invasion by inhibiting cyclin B/Cdk1 and other promotility proteins

“…However, as we have reported previously (Toby et al, 2003), even moderate overexpression of HEI10 in mammalian cells leads to accumulation of most of the protein in apparently aggregated structures associated with the Golgi compartment, making interpretation of such an experiment problematic. In lieu of being able to readily overexpress HEI10, we have studied the distribution of HEI10 in different tissues and cell types, to determine if HEI10 expression levels suggest a characteristic profile of migratory or invasive behavior (Supplementary Figure S1).…”

“…Following siRNA transfection of U20S or MCF7 cells, HEI10 levels were typically reduced by B75% for up to 5 days (Figure 1a-c). As HEI10 binds and promotes degradation of cyclin B (Toby et al, 2003), we first assessed whether HEI10 depletion induced proliferation defects by measuring cell doubling. For the first 2 days after transfection, both HEI10-depleted and control cells appeared similar (not shown).…”

“…Human enhancer of invasion, clone 10 (HEI10) was first identified by our group in a functional genomic screen for novel human genes that influenced cell cycle progression and/or polarization (Toby et al, 2003). Overexpression of HEI10 causes yeast to extend the G2 phase of cell cycle and to become hyperpolarized.…”