Cyclin A and Cks1 promote kinase consensus switching to non-proline-directed CDK1 phosphorylation.

Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alter substrate choice, with intermediate- and low-sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence is shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 enhance non-proline-directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell-cycle-regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline-directed CDK1 sites.

Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation.

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

Differences in the Conformational Energy Landscape of CDK1 and CDK2 Suggest a Mechanism for Achieving Selective CDK Inhibition.

Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.

Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein.

BACKGROUND: Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. METHODS: We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. RESULTS: We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4me3 mark at transcriptional start sites. CONCLUSIONS: Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.

Cyclin-Dependent Kinase (CDK) Inhibitors: Structure-Activity Relationships and Insights into the CDK-2 Selectivity of 6-Substituted 2-Arylaminopurines.

Purines and related heterocycles substituted at C-2 with 4'-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favor competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency toward CDK2 (IC50 0.044 µM) but was ∼2000-fold less active toward CDK1 (IC50 86 µM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket and that is preferred in CDK2 but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.

CDK1 structures reveal conserved and unique features of the essential cell cycle CDK.

CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

Oxidative stress, gene expression, and protein changes induced in the human placenta during labor.

Malperfusion of the placenta has been implicated as a cause of oxidative stress in complications of human pregnancy, leading to release of proinflammatory cytokines and anti-angiogenic factors into the maternal circulation. Uterine contractions during labor are known to be associated with intermittent utero-placental perfusion. We therefore tested whether oxidative stress, proinflammatory cytokines, and angiogenic regulators were increased in placentas subjected to short (<5 hours) and long (>15 hours) labor compared with nonlabored controls delivered by cesarean section. In addition, broader changes in gene transcripts were assessed by microarray analysis. Oxidative stress, activation of the nuclear factor-kappaB pathway, tumor necrosis factor-alpha and interleukin 1beta all increased in placental tissues after labor. Stabilization of hypoxia-inducible factor-1alpha and increased vascular endothelial growth factor soluble receptor-1 were also observed. By contrast, tissue levels of placenta growth factor decreased. Apoptosis was also activated in labored placentas. The magnitude of these changes related to the duration of labor. After labor, 55 gene transcripts were up-regulated and 35 down-regulated, and many of these changes were reflected at the protein level. In conclusion, labor is a powerful inducer of placental oxidative stress, inflammatory cytokines, and angiogenic regulators. Our findings are consistent with intermittent perfusion being the initiating cause. Placentas subjected to labor do not reflect the normal in vivo state at the molecular level.

Endoplasmic reticulum stress exacerbates ischemia-reperfusion-induced apoptosis through attenuation of Akt protein synthesis in human choriocarcinoma cells.

Oxidative stress is central to ischemia-reperfusion injury. The role of the endoplasmic reticulum (ER) in this process is uncertain. In ER signaling, PERK-Nrf2 and Ire-CHOP are two pathways that determine cell fate under stress. PERK-Nrf2 up-regulates antioxidant enzyme expression whereas Ire-CHOP promotes apoptosis. We have identified a novel pathway in ER stress-induced apoptosis after ischemia-reperfusion in vitro involving translational suppression of the survival kinase PKB/Akt (Akt), and elucidated an alternative protective role of antioxidants in the regulation of Akt activity. Using human choriocarcinoma JEG-3 cells, we found that sustained activation of ER stress by tunicamycin or thapsigargin exacerbated apoptosis in oxygen-glucose-deprived cells during reoxygenation. This was mediated via a reduction in phosphorylated Akt secondary to down-regulation of protein translation rather than suppression of phosphorylation. Transient overexpression of wild-type Akt, but not kinase-dead Akt, in JEG-3 cells diminished tunicamycin-OGD reoxygenation-induced apoptosis. The antioxidants Trolox and Edaravone reduced apoptosis, but the protective effect of Trolox was abrogated by the PI3K inhibitor, LY294002. We speculate that sustained ER stress may contribute to the placental dysfunction seen in human pregnancy complications.