Proteolysis Of Mitotic Cyclins Research Articles

Abstract PR11: APC/CCdh1 maintains primordial follicles, germinal vesicle arrest and ensures balanced segregation of chromosomes by enabling removal of Shugoshin-2 from chromosomes arms

Abstract Germ cells in mammals, including humans, remain arrested at the primordial follicle stage from a few days to many years depending on the species. At each hormonal cycle, a few of these cells grow in size to become fully-grown oocytes but remain arrested in meiotic prophase. A resumption of meiosis is heralded by germinal vesicle breakdown (GVBD), a gradual rise in Cdk1 activity, condensation of bivalent chromosomes, and their eventual alignment on metaphase plates with maternal and paternal kinetochore pairs pulled in opposite directions (co-orientation). The first meiotic division is triggered by activation of a form of the Anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20), which activates separase and thereby destroys sister chromatid cohesion along chromosome arms, resolving chiasmata. Because cohesion around centromeres is protected from separase by protein phosphatase 2A (PP2A) recruited by shugoshin (Sgol2), bivalents are converted at this division to dyad chromosomes in which sister chromatids are held together by peri-centric cohesin. We show here that an alternative version of the APC/C associated with the Cdc20-like Cdh1 protein (APC/CCdh1) has equally important roles. By promoting proteolysis of mitotic cyclins and Cdc25B, APC/CCdh1 delays the increase in Cdk1 activity leading to GVBD. More surprising, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of Sgol2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata. Lastly, by targeting proteins other than cyclins, APC/CCdh1 prevents apoptosis of primordial follicles during their extended prophase (G2) arrest. This abstract is also being presented as Poster A24. Citation Format: Ahmed Rattani, Randy Ballestros Mejia, Katherine Roberts, Maurici B Roig, Jonathan Godwin, Manuel Eguren, Sugako Ogushi, Elwy Okaz, Alberto Pendas, Malumbres Marcos, Mary Herbert, Kim Nasmyth. APC/CCdh1 maintains primordial follicles, germinal vesicle arrest and ensures balanced segregation of chromosomes by enabling removal of Shugoshin-2 from chromosomes arms. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR11.

A Mathematical Model of Mitotic Exit in Budding Yeast: The Role of Polo Kinase

Cell cycle progression in eukaryotes is regulated by periodic activation and inactivation of a family of cyclin–dependent kinases (Cdk's). Entry into mitosis requires phosphorylation of many proteins targeted by mitotic Cdk, and exit from mitosis requires proteolysis of mitotic cyclins and dephosphorylation of their targeted proteins. Mitotic exit in budding yeast is known to involve the interplay of mitotic kinases (Cdk and Polo kinases) and phosphatases (Cdc55/PP2A and Cdc14), as well as the action of the anaphase promoting complex (APC) in degrading specific proteins in anaphase and telophase. To understand the intricacies of this mechanism, we propose a mathematical model for the molecular events during mitotic exit in budding yeast. The model captures the dynamics of this network in wild-type yeast cells and 110 mutant strains. The model clarifies the roles of Polo-like kinase (Cdc5) in the Cdc14 early anaphase release pathway and in the G-protein regulated mitotic exit network.

Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast

Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Delta strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Delta cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Delta and hsl1Delta resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

Mitotic degradation of cyclin A is mediated by multiple and novel destruction signals.

Exit from mitosis requires Cdk1 inactivation, with the most prominent mechanism of Cdk1 inactivation being proteolysis of mitotic cyclins [1]. In higher eukaryotes this involves sequential destruction of A- and B-type cyclins. CycA is destroyed first, and CycA/Cdk1 inactivation is required for the metaphase-to-anaphase transition [2]. The degradation of CycA is delayed in response to DNA damage but is not prevented when the spindle checkpoint is activated [3, 4]. Cyclin destruction is thought to be mediated by a conserved motif, the destruction box (D box). Like B-type cyclins, A-type cyclins contain putative destruction box sequences in their N termini [5]. However, no detailed in vivo analysis of the sequence requirements for CycA destruction has been described so far. Here we tested several mutations in the CycA coding region for destruction in Drosophila embryos. We show that D box sequences are not essential for mitotic destruction of CycA. Destruction is mediated by at least three different elements that act in an overlapping fashion to mediate its mitotic degradation.

Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.

Mos‐induced p42 mitogen‐activated protein kinase activation stabilizes M‐phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B Cdc2 ), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.