The centrosomal protein RAS association domain family protein 1A (RASSF1A)-binding protein 1 regulates mitotic progression by recruiting RASSF1A to spindle poles

The protein RAS association domain family protein 1A (RASSF1A), which is encoded by a gene that is frequently silenced in many types of sporadic tumor, functions in mitosis as a regulator of the anaphase-promoting complex (APC). With the use of a yeast two-hybrid screen, we identified a human protein, previously designated C19ORF5, that interacts with RASSF1A. This protein, here redesignated RASSF1A-binding protein 1 (RABP1), contains two microtubule-associated protein domains, and its association with RASSF1A was confirmed in mammalian cells by immunoprecipitation and immunofluorescence analyses. RABP1 was found to be localized to the centrosome throughout the cell cycle in a manner dependent on its microtubule-associated protein domains. Ectopic expression of RABP1 induced both stabilization of mitotic cyclins and mitotic arrest at prometaphase in a RASSF1A-dependent manner. It also increased the extent of association between RASSF1A and Cdc20. Conversely depletion of RABP1 by RNA interference prevented both the localization of RASSF1A to the spindle poles as well as its binding to Cdc20, resulting in premature destruction of mitotic cyclins and acceleration of mitotic progression. These findings indicate that RABP1 is required for the recruitment of RASSF1A to the spindle poles and for its inhibition of APC-Cdc20 activity during mitosis.     

Control of APC-Cdc20 by the Tumor Suppressor RASSF1A

The tumor suppressor gene RASSF1A is frequently hypermethylated in various tumors. However, how RASSF1A functions in tumor suppression was unknown. Our study shows that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression by inhibiting APC-Cdc20. RASSF1A appears to work in early pro-metaphase, after Emi1 destruction and before activation of the Mad2-dependent spindle checkpoint. Loss of RASSF1A expression induces premature APC activation, thereby resulting in acceleration of mitotic cyclin degradation and mitotic progression as well as induction of mitotic abnormalities. Thus, RASSF1A plays a critical role in mitotic progression and tumor suppression by controlling APC-Cdc20 activity.     

Control of APC-Cdc20 by the tumor suppressor RASSF1A

The tumor suppressor gene RASSF1A is frequently hypermethylated in various tumors. However, how RASSF1A functions in tumor suppression was unknown. Our study shows that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression by inhibiting APC-Cdc20. RASSF1A appears to work in early pro-metaphase, after Emi1 destruction and before activation of the Mad2-dependent spindle checkpoint. Loss of RASSF1A expression induces premature APC activation, thereby resulting in acceleration of mitotic cyclin degradation and mitotic progression as well as induction of mitotic abnormalities. Thus, RASSF1A plays a critical role in mitotic progression and tumor suppression by controlling APC-Cdc20 activity.     

The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex

The tumour suppressor gene RASSF1A is frequently silenced in lung cancer and other sporadic tumours as a result of hypermethylation of a CpG island in its promoter. However, the precise mechanism by which RASSF1A functions in cell cycle regulation and tumour suppression has remained unknown. Here we show that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression. RASSF1A localizes to microtubules during interphase and to centrosomes and the spindle during mitosis. The overexpression of RASSF1A induced stabilization of mitotic cyclins and mitotic arrest at prometaphase. RASSF1A interacts with Cdc20, an activator of the anaphase-promoting complex (APC), resulting in the inhibition of APC activity. Although RASSF1A does not contribute to either the Mad2-dependent spindle assembly checkpoint or the function of Emi1 (ref. 1), depletion of RASSF1A by RNA interference accelerated the mitotic cyclin degradation and mitotic progression as a result of premature APC activation. It also caused a cell division defect characterized by centrosome abnormalities and multipolar spindles. These findings implicate RASSF1A in the regulation of both APC-Cdc20 activity and mitotic progression.     

The RASSF1A tumor suppressor restrains anaphase-promoting complex/cyclosome activity during the G1/S phase transition to promote cell cycle progression in human epithelial cells

Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.     

Cullin-4A·DNA damage-binding protein 1 E3 ligase complex targets tumor suppressor RASSF1A for degradation during mitosis

Tumor suppressor RASSF1A (RAS association domain family 1, isoform A) is known to play an important role in regulation of mitosis; however, little is known about how RASSF1A is regulated during the mitotic phase of the cell cycle. In the present study, we have identified Cullin-4A (CUL4A) as a novel E3 ligase for RASSF1A. Our results demonstrate that DNA damage-binding protein 1 (DDB1) functions as a substrate adaptor that directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex. Depletion of DDB1 also diminishes intracellular interactions between RASSF1A and CUL4A. Our results also show that RASSF1A interacts with DDB1 via a region containing amino acids 165-200, and deletion of this region abolishes RASSF1A and DDB1 interactions. We have found that CUL4A depletion results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of CUL4A and DDB1 markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We further show that CUL4A-mediated RASSF1A degradation occurs during mitosis, and depletion of CUL4A markedly reverses mitotic-phase-stimulated RASSF1A degradation. We also note that overexpression of CUL4A antagonizes the ability of RASSF1A to induce M-phase cell cycle arrest. Thus, our present study demonstrates that the CUL4A·DDB1 E3 complex is important for regulation of RASSF1A during mitosis, and it may contribute to inactivation of RASSF1A and promoting cell cycle progression.     

Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity

The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity.     

Cdk1-phosphorylated CUEDC2 promotes spindle checkpoint inactivation and chromosomal instability

Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C-Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results suggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability.     

Cdc20: a WD40 activator for a cell cycle degradation machine

Cdc20 is an essential cell-cycle regulator required for the completion of mitosis in organisms from yeast to man and contains at its C terminus a WD40 repeat domain that mediates protein-protein interactions. In mitosis, Cdc20 binds to and activates the ubiquitin ligase activity of a large molecular machine called the anaphase-promoting complex/cyclosome (APC/C) and enables the ubiquitination and degradation of securin and cyclin B, thus promoting the onset of anaphase and mitotic exit. APC/C(Cdc20) is temporally and spatially regulated during the somatic and embryonic cell cycle by numerous mechanisms, including the spindle checkpoint and the cytostatic factor (CSF). Therefore, Cdc20 serves as an integrator of multiple intracellular signaling cascades that regulate progression through mitosis. This review summarizes recent progress toward the understanding of the functions of Cdc20, the mechanisms by which it activates APC/C, and its regulation by phosphorylation and by association with its binding proteins.     

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.     

Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl‐1 during an extended mitosis requires the anaphase‐promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCF^Fbw7. Using live‐cell imaging, we show that the loss of Mcl‐1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine‐arginine (IR) C‐terminal tail regulates the manner in which Mcl‐1 engages with the APC/C, converting Mcl‐1 from a Cdc20‐dependent and checkpoint‐controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl‐1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl‐1 degradation, providing an improved strategy to kill cancer cells.     

RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1

ABSTRACT: BACKGROUND: RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). METHODS: Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. RESULTS: The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. CONCLUSIONS: Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.     

APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.     

RASSF7 is a member of a new family of RAS association domain-containing proteins and is required for completing mitosis

Mitosis is a fundamental feature of all cellular organisms. It must be tightly regulated to allow normal tissue growth and to prevent cancer formation. Here, we identify a new protein that is required for mitosis. We show that the Ras association (RA) domain-containing protein, RASSF7, is part of an evolutionarily conserved group of four proteins. These are RASSF7, RASSF8, and two new RASSF proteins P-CIP1/RASSF9 and RASSF10. We call this group the N-terminal RASSF family. We analyzed the function of Xenopus RASSF7. RASSF7 was found to be expressed in several embryonic tissues including the skin, eyes, and neural tube. Knocking down its function led to cells failing to form a mitotic spindle and arresting in mitosis. This caused nuclear breakdown, apoptosis, and a striking loss of tissue architecture in the neural tube. Consistent with a role in spindle formation, RASSF7 protein was found to localize to the centrosome. This localization occurred in a microtubule-dependent manner, demonstrating that there is a mutually dependant relationship between RASSF7 localization and spindle formation. Thus RASSF7, the first member of the N-terminal RASSF family to be functionally analyzed, is a centrosome-associated protein required to form a spindle and complete mitosis in the neural tube.     

Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C

Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.