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RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/CCdc20 is the master switch and regulator of mitosis. The activity of APC/CCdc20 is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/CCdc20 during mitosis. Our study showed that RASSF1A and APC/CCdc20 form a molecular relay that regulates the APC/CCdc20 activity at early mitosis. We found that RASSF1A inhibits APC/CCdc20 function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/CCdc20 in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/CCdc20. Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/CCdc20 during the course of mitotic progression. As a result of RASSF1A degradation, APC/CCdc20 can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A-APC/CCdc20 interaction in ensuring the sequential progression of mitosis. [ABSTRACT FROM AUTHOR]

The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1–Cul1–F–box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1–S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D–cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1–S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival.Oncogene (2008) 27, 3176–3185; doi:10.1038/sj.onc.1210971; published online 10 December 2007 [ABSTRACT FROM AUTHOR]

Here we report the genomic cloning and characterization of the murine A1 genes, which belong to the bcl-2 gene family. Southern analysis indicated the existence of at least four A1 genes in the murine genome and four different A1 genes, designated A1-a, -b, -c and -d, were cloned from the murine genomic library. The A1-a, -b and -d genes consisted of two exons, whereas the A1-c gene contained 1 bp insertion in the coding region which may result in an aberrant and truncated protein by frame-shift. With the exception of A1-c, the coding regions among A1 genes are highly conserved at >97% at the nucleotide level and at >96% at the amino acid level. A1-a, -b and -d genes appeared to be expressed specifically in organs containing many neutrophils. In neutrophils, A1-a, -b and -d transcripts were detected at a comparable level. Our data suggest that the multiple A1 genes in mice were generated by gene duplication and each of them may function as anti-apoptotic molecules in neutrophils. [ABSTRACT FROM PUBLISHER]

The U box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition., (Copyright 2001 Academic Press.)

The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCF(Skp2). The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed., (Copyright 2001 Academic Press.)