Metabolic activity is a crucial determinant of a cell's decision to proliferate or die. Although it is not fully understood how metabolic pathways such as glycolysis and the pentose phosphate pathway ...communicate to cell cycle and apoptotic effectors, it is clear that a complex network of signalling molecules is required to integrate metabolic inputs. D-type cyclins, cyclin-dependent kinases, the anaphase-promoting complex, p53, caspase 2 and B cell lymphoma 2 proteins, among others, have been shown to be regulated by metabolic crosstalk. Elucidating these pathways is of great importance, as metabolic aberrations and their downstream effects are known to contribute to the aetiology of cancer and degenerative disorders.
Apoptosis, a form of programmed cell death, is executed by a family of zymogenic proteases known as caspases, which cleave an array of intracellular substrates in the dying cell. Many proapoptotic ...stimuli trigger cytochrome c release from mitochondria, promoting the formation of a complex between Apaf-1 and caspase-9 in a caspase-activating structure known as the apoptosome. In this review, we describe knockout and knockin studies of apoptosome components, elegant structural and biochemical experiments, and analyses of the apoptosome in various cancers and other disease states, all of which have provided new insight into this critical locus of apoptotic control.
Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert ...injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.
Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic ...kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1-PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.
The complex process of apoptosis is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities. Accumulating evidence suggests that cell death pathways are finely ...tuned by multiple signaling events, including direct phosphorylation of caspases, whereas kinases are often substrates of active caspases. Importantly, caspase-mediated cleavage of kinases can terminate prosurvival signaling or generate proapoptotic peptide fragments that help to execute the death program and facilitate packaging of the dying cells. Here, we review caspases as kinase substrates and kinases as caspase substrates and discuss how the balance between cell survival and cell death can be shifted through crosstalk between these two enzyme families.
Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and ...p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.
Ataxia telangiectasia mutant (ATM) is an S/T-Q–directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited ...by the MRN protein complex meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.
Movement through the cell cycle is controlled by the temporally and spatially ordered activation of cyclin-dependent kinases paired with their respective cyclin binding partners. Cell cycle events ...occur in a stepwise fashion and are monitored by molecular surveillance systems to ensure that each cell cycle process is appropriately completed before subsequent events are initiated. Cells prevent entry into mitosis while DNA replication is ongoing, or if DNA is damaged, via checkpoint mechanisms that inhibit the activators and activate the inhibitors of mitosis, Cdc25 and Wee1, respectively. Once DNA replication has been faithfully completed, Cdc2/Cyclin B is swiftly activated for a timely transition from interphase into mitosis. This sharp transition is propagated through both positive and negative feedback loops that impinge upon Cdc25 and Wee1 to ensure that Cdc2/Cyclin B is fully activated. Recent reports from a number of laboratories have revealed a remarkably complex network of kinases and phosphatases that coordinately control Cdc25 and Wee1, thereby precisely regulating the transition into mitosis. Although not all factors that inhibit Cdc25 have been shown to activate Wee1 and vice versa, a number of regulatory modules are clearly shared in common. Thus, studies on either the Cdc25 or Wee1-regulatory arm of the mitotic control pathway should continue to shed light on how both arms are coordinated to smoothly regulate mitotic entry.
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