Cul1, a member of the cullin ubiquitin ligase family, forms a multiprotein complex known as SCF and plays an essential role in numerous cellular and biological activities. A Cul1 homologue, p185 (Cul7), has been isolated as an simian virus 40 large T antigen-binding protein. To understand the physiological role of p185, we generated mice lacking p185. p185 ؊/؊ embryos are runted and die immediately after birth because of respiratory distress. Dermal and hypodermal hemorrhage is detected in mutant embryos at late gestational stage. p185 ؊/؊ placentas show defects in the differentiation of the trophoblast lineage with an abnormal vascular structure. We demonstrate that p185 forms an SCF-like complex with Skp1, Rbx1, Fbw6 (Fbx29), and FAP68 (FAP48, glomulin). FAP68 has recently been identified as a gene responsible for familial glomuvenous malformation. These results suggest that p185 forms a multiprotein complex and plays an important role in vascular morphogenesis.T he rapid destruction of regulatory proteins by ubiquitinmediated proteolysis plays an important role in various biological processes. Protein ubiquitination is carried out by the sequential action of three enzymes, namely E1, E2, and E3 (1). E3 ubiquitin ligases determine the specificity of the substrate protein, and it is likely that protein ubiquitination in vivo is controlled primarily by regulating E3 activity or E3-substrate interaction (2). The Skp1-Cul1-F-box (SCF) complex is a well characterized E3 ubiquitin ligase that plays an essential role in a wide variety of activities, including cell cycle control (3, 4), muscle atrophy (5, 6), and protein quality control (7). To date, the human cullin protein family consists of six members: CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5 (8). Although the physiological function of each cullin protein is largely unknown, mouse knockout evidence has demonstrated their biological significance. Cul1-, Cul3-, and Cul4A-deficient mice show early embryonic lethality (9-12), indicating their crucial role in early mammalian development.F box proteins are the variable subunits of the SCF complex, and a large number of human F box proteins have been reported (13,14). F box proteins contain an F box motif that binds to Skp1 and a carboxyl-terminal domain that specifies association with substrate proteins (15,16). SCF targets a number of different substrate proteins by associating with specific F box proteins (16,17). Among the human cullin proteins, SKP1 protein has been reported to bind specifically to CUL1 (18). Degradation of some F box proteins, such as Cdc4, Grr1, and SKP2 (22) in humans, are mediated by the SCF complex itself.Simian virus 40 large T antigen transforms a variety of rodent cells in vitro and in vivo. p185 (KIAA0076, p193, Cul7) was isolated as an simian virus 40 large T antigen-associated protein in murine cells (23,24). Although it has been reported that overexpression of p185 induces apoptosis in NIH 3T3 cells (24), the biological and molecular functions of p185 remain unclear. Here we demonstrate ...

Simian virus 40 large T antigen (TAg) is a viral oncoprotein that can promote cellular transformation. TAg's transforming activity results in part by binding and inactivating key tumor suppressors, including p53 and the retinoblastoma protein (pRb). We have identified a TAg-associated 185-kDa protein that has significant homology to the cullin family of E3 ubiquitin ligases. TAg binds to an SCF-like complex that contains p185/Cul7, Rbx1, and the F box protein Fbw6. This SCF-like complex binds to an N-terminal region of TAg. Several p185/Cul7-binding-deficient mutants of TAg were generated that retained binding to pRb and p53 and were capable of overcoming Rb-mediated repression of E2F transcription. Despite binding to pRb and p53, these p185/Cul7-binding-defective mutants of TAg were unable to transform primary mouse embryo fibroblasts. Cells expressing p185/Cul7-binding-defective mutants of TAg were unable to grow to high density or grow in an anchorage-independent manner as determined by growth in soft agar. Considering the significance of other TAg-interacting proteins in regulation of the cell cycle, p185/Cul7 may also regulate an important growth control pathway.

SUMMARY Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here we show that Glomulin (Glmn), a protein found mutated in the vascular disorder Glomuvenous Malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity indicating that Glmn modulates the E3 activity of CRL1Fbw7. These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.