In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin-cyclindependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes involved in nutrient homeostasis. We isolated myo-D-inositol heptakisphosphate (IP 7 ) as a cellular component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP 7 is necessary for Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP 7 increased upon phosphate starvation, and yeast mutants defective in IP 7 production failed to inhibit Pho80-Pho85 in response to phosphate starvation. These observations reveal regulation of a cyclin-CDK complex by a metabolite and suggest that a complex metabolic network mediates signaling of phosphate availability.In response to nutrient limitation, cells increase transcription of starvation response genes. Many proteins that function in nutrient response systems have been identified, but the molecular details of their regulation are not well understood (1). The Saccharomyces cerevisiae Pho80-Pho85 cyclin-CDK complex is a key regulator in the phosphate (P i )-responsive (PHO) signaling pathway (2,3). When cells are grown in medium that is rich in P i , Pho80-Pho85 is active and phosphorylates the transcription factor Pho4 (4), resulting in its export from the nucleus (5). Upon P i starvation, Pho80-Pho85 kinase activity is decreased, leading to nuclear accumulation of Pho4 and transcription of PHO genes. In vivo, the Pho81 CDK inhibitor (CKI) is bound to Pho80-Pho85 regardless of P i conditions and is required for the inhibition of Pho80-Pho85 activity in response to P i -limitation (6).We used a biochemical strategy to identify cellular components that influence Pho80-Pho85-Pho81 activity. The Pho80-Pho85-Pho81 complex immunopurified from cells grown in P i -rich conditions actively phosphorylated Pho4 in vitro, whereas the complex isolated from P i -starved cells was less active (6) (Fig. 1A). To identify regulators of Pho80-Pho85-Pho81, we added extracts from cells grown in P i -rich and P i -starved conditions to active (from P i -rich) and inactive (from P i -starved) immunopurified Pho80-Pho85-Pho81 complexes and performed kinase assays with recombinant Pho4 serving as the substrate. Extracts from P i -starved cells efficiently inactivated Pho80-Pho85-Pho81, whereas the P i -rich cell extract had little effect (Fig. 1B). When the extract of P i -starved cells was fractionated into fractions of large (>3 kD) and small (<3 kD) molecular size, most inhibitory activity was found in the fractions of small molecular size. This activity had little effect on Pho80-Pho85 purified from pho81Δ cells (Fig. 1C), indicating that, as is true in vivo (6), the inhibitory activity requires Pho81 for its function.

Pyruvate kinase isoform M2 (PKM2) plays an important role in the growth and metabolic reprogramming of cancer cells in stress conditions. Here, we report that SAICAR (succinylaminoimidazolecarboxamide ribose-5′-phosphate, an intermediate of the de novo purine nucleotide synthesis pathway) specifically stimulates PKM2. Upon glucose starvation, cellular SAICAR concentration increases in an oscillatory manner and stimulates PKM2 activity in cancer cells. Changes in SAICAR levels in cancer cells alter cellular energy level, glucose uptake, and lactate production. The SAICAR-PKM2 interaction also promotes cancer cell survival in glucose-limited conditions. SAICAR accumulation is not observed in normal adult epithelial cells or lung fibroblasts regardless of glucose conditions. This allosteric regulation may explain how cancer cells coordinate different metabolic pathways to optimize their growth in the nutrient-limited conditions commonly observed in the tumor microenvironment.

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified foursubunit Pol ζ4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3′ guanine and Pol ζ4 to extend the primers., composed of the catalytic Rev3 and accessary Rev7 subunits, is an error-prone DNA translesion polymerase that causes both spontaneous and DNA damage-induced mutagenesis (1, 2). More than two-thirds of the 1,504 residues in yeast Rev3 share sequence homology with all B-family DNA polymerases, including Pols α, δ, and e, which are responsible for the bulk of high-fidelity genomic replication in eukaryotes (3). Unlike the typical B-family polymerases, Pol ζ lacks an intrinsic 3′-5′ exonuclease activity and thus has no proofreading function (2). Human homologs of REV3 (REV3L) and REV7 (MAD2L2; hereafter referred to as REV7) genes were identified shortly after yeast Pol ζ was characterized. Human Rev3 contains 3,130 residues and is twice as large as the yeast counterpart (4). Human and yeast Rev7 are homologous (5) and bear sequence similarity to the mitotic checkpoint proteins Mad2 (6). Unlike Saccharomyces cerevisiae REV3 and REV7 genes, which are nonessential and whose knockout leads only to a decreased rate of damage-induced mutagenesis (7, 8), Rev3l knockout in mice is embryonic-lethal (9), and mouse Rev3l −/− embryonic stem cells are not viable (10, 11). Human and mouse cell cultures obtained from conditional Rev3l knockout show genome instability and growth defects without an external challenge of DNA damage (12)(13)(14). DNA pol ζ is apparently essential for normal cell proliferation and embryogenesis in mammals.Translesion synthesis (TLS) and DNA-damage-induced mutagenesis are the best-characterized functions of Pol ζ. Absence of the yeast REV3 gene leads to sensitivity to UV light and intrastrand and interstrand cross-linking agents (2, 15). DNA Pol ζ has been shown to induce multiple base substitutions as well as more complex mutations in yeast (7,16,17) and may contribute to hypermutation in Ig genes in mammals (18,19). The TLS function of DNA Pol ζ has been implicated in its role of mediating resistance to platinum-based chemotherapies (20)(21)(22). Owing to the conservation of B-f...

When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.

Abnormal metabolism and sustained proliferation are hallmarks of cancer. Pyruvate kinase M2 (PKM2) is a metabolic enzyme that plays important roles in both processes. Recently, PKM2 was shown to have protein kinase activity phosphorylating histone H3 and promoting cancer cell proliferation. However, the mechanism and extent of this novel protein kinase in cancer cells remain unclear. Here, we report that binding of SAICAR, a metabolite abundant in proliferating cells, induces PKM2’s protein kinase activity in vitro and in cells. Protein microarray experiments revealed that more than 100 human proteins– mostly protein kinases– are phosphorylated by PKM2-SAICAR. In particular, PKM2-SAICAR phosphorylates and activates Erk1/2, which in turn sensitizes PKM2 for SAICAR-binding through phosphorylation. Additionally, PKM2-SAICAR was necessary to induce sustained Erk1/2 activation and mitogen-induced cell proliferation. Thus, the ligand-induced protein kinase activity from PKM2 is a mechanism that directly couples cell proliferation with intracellular metabolic status.

Endothelial progenitor cells (EPCs) act as endothelial precursors that promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. These facts prompt the hypothesis that EPC transplantation should accelerate the wound-repair process by facilitating neovascularization and the production of various molecules related to wound healing. In a murine dermal excisional wound model, EPC transplantation accelerated wound re-epithelialization compared with the transplantation of mature endothelial cells (ECs) in control mice. When the wounds were analyzed immunohistochemically, the EPCtransplanted group exhibited significantly more monocytes/ macrophages in the wound at day 5 after injury than did the EC-transplanted group. This observation is consistent with enzyme-linked immunosorbent assay results showing that EPCs produced in abundance several chemoattractants of monocytes and macrophages that are known to play a pivotal role in the early phase of wound healing. At day 14 after injury, the EPC-transplanted group showed a statistically significant increase in vascular density in the granulation tissue relative to that of the EC-transplanted group. Fluorescence microscopy revealed that EPCs preferentially moved into the wound and were directly incorporated into newly formed capillaries in the granulation tissue. These results suggest that EPC transplantation will be useful in dermal wound repair and skin regeneration, because EPCs both promote the recruitment of monocytes/macrophages into the wound and increase neovascularization. Stem Cells 2005;23:1571-1578

The human DNA polymerase gamma (Pol c) is responsible for DNA replication in mitochondria. Pol c is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol c-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol c active site almost identically to the substrate dCTP, providing a structural basis for Pol c-mediated drug toxicity. When compared to the apo form, Pol c undergoes intra-and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol cB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol c mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol c, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol c-mediated drug toxicity.

Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η− cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.cancer therapy | monofunctional platinum drug candidates | pol eta | X-ray crystallography