Farida Tripodi

An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes.

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.

The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae

The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Δcka2-8ts strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34S130AS167A mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2ts yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.

Cell growth and cell cycle In Saccharomyces cerevisiae: basic regulatory design and protein-protein interaction network

In this review we summarize the major connections between cell growth and cell cycle in the model eukaryote Saccharomyces cerevisiae. In S. cerevisiae regulation of cell cycle progression is achieved predominantly during a narrow interval in the late G1 phase known as START (Pringle and Hartwell, 1981). At START a yeast cell integrates environmental and internal signals (such as nutrient availability, presence of pheromone, attainment of a critical size, status of the metabolic machinery) and decides whether to enter a new cell cycle or to undertake an alternative developmental program. Several signaling pathways, that act to connect the nutritional status to cellular actions, are briefly outlined. A Growth & Cycle interaction network has been manually curated. More than one fifth of the edges within the Growth & Cycle network connect Growth and Cycle proteins, indicating a strong interconnection between the processes of cell growth and cell cycle. The backbone of the Growth & Cycle network is composed of middle-degree nodes suggesting that it shares some properties with HOT networks. The development of multi-scale modeling and simulation analysis will help to elucidate relevant central features of growth and cycle as well as to identify their system-level properties. Confident collaborative efforts involving different expertises will allow to construct consensus, integrated models effectively linking the processes of cell growth and cell cycle, ultimately contributing to shed more light also on diseases in which an altered proliferation ability is observed, such as cancer.

Post-translational modifications on yeast carbon metabolism: Regulatory mechanisms beyond transcriptional control

BACKGROUND Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and environmental perturbations. This can be achieved by a massive reprogramming of gene expression. However, the transcriptional response cannot explain the complexity of metabolic regulation, and mRNA stability regulation, non-covalent binding of allosteric effectors and post-translational modifications of enzymes (such as phosphorylation, acetylation and ubiquitination) are also involved, especially as short term responses, all converging in modulating enzyme activity. SCOPE OF REVIEW The functional significance of post-translational modifications (PTMs) to the regulation of the central carbon metabolism is the subject of this review. MAJOR CONCLUSIONS A genome wide analysis of PTMs indicates that several metabolic enzymes are subjected to multiple PTMs, suggesting that yeast cells can use different modifications and/or combinations of them to specifically respond to environmental changes. Glycolysis and fermentation are the pathways where phosphorylation, acetylation and ubiquitination are most frequent, while enzymes of storage carbohydrate metabolism are especially phosphorylated. Interestingly, some enzymes, such as the 6-phosphofructo-2-kinase Pfk26, the phosphofructokinases Pfk1 and Pfk2 and the pyruvate kinase Cdc19, are hubs of PTMs, thus representing central key regulation nodes. For the functionally better characterized enzymes, the role of phosphorylations and lysine modifications is discussed. GENERAL SIGNIFICANCE This review focuses on the regulatory mechanisms of yeast carbon metabolism, highlighting the requirement of quantitative, systematical studies to better understand PTM contribution to metabolic regulation.

Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate-activated protein kinase and induction of apoptosis

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-trans-20, (±)-trans-21, and enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC(50) values of 3-13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymerization and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase (AMPK), activation of caspase-3, and induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer.

Dual action antifungal small molecule modulates multidrug efflux and TOR signaling

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. We establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for fungal infectious disease that evades resistance.

CK2 activity is modulated by growth rate in Saccharomyces cerevisiae.

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and k(cat). Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.

Snf1/AMPK promotes SBF and MBF-dependent transcription in budding yeast

Snf1, the yeast AMP-activated kinase homolog, regulates the expression of several genes involved in adaptation to glucose limitation and in response to cellular stresses. We previously demonstrated that Snf1 interacts with Swi6, the regulatory subunit of SBF and MBF complexes, and activates CLB5 transcription. Here we report that, in α-factor synchronized cells in 2% glucose, the loss of the Snf1 catalytic subunit impairs the binding of SBF and MBF complexes and the subsequent recruitment of the FACT complex and RNA Polymerase II to promoters of G1-genes. By using an analog-sensitive allele of SNF1, SNF1(as)(I132G), encoding a protein whose catalytic activity is selectively inhibited in vivo by 2-naphthylmethyl pyrazolopyrimidine 1, we show that the inhibition of Snf1 catalytic activity affects the expression of G1-genes causing a delayed entrance into S phase in cells synchronized in G1 phase by α-factor treatment or by elutriation. Moreover, Snf1 is detected in immune complexes of Rpb1, the large subunit of RNA Polymerase II, and is present at both promoters and coding regions of SBF- and MBF-regulated genes 20min after α-factor release, suggesting a direct role for Snf1 in the activation of the G1-regulon transcription.

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae

Background: The Snf1/AMPK and PKA pathways are crucial for nutrient sensing and utilization in yeast. Results: A novel cross-talk mechanism between Snf1/AMPK and PKA is proposed. Conclusion: Snf1 and Cyr1 interact in a nutrient-independent manner. Active Snf1 phosphorylates Cyr1 and negatively regulates cAMP content and PKA-dependent transcription. Significance: This is the first evidence of regulation of PKA pathway by Snf1/AMPK. In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription.

Nutritional modulation of CK2 in Saccharomyces cerevisiae: regulating the activity of a constitutive enzyme.

CK2 is a highly conserved protein kinase involved in different cellular processes, which shows a higher activity in actively proliferating mammalian cells and in various types of cancer and cancer cell lines. We recently demonstrated that CK2 activity is strongly influenced by growth rate in yeast cells as well. Here, we extend our previous findings and show that, in cells grown in either glucose or ethanol-supplemented media, CK2 presents no alteration in Km for both the ATP and the peptide substrate RRRADDSDDDDD, while a significant increase in Vmax is observed. In chemostat-grown cells, no difference of CK2 activity was observed in cells grown at the same dilution rate in media supplemented with either ethanol or glucose, excluding the contribution of carbon metabolism on CK2 activity. By using the eIF2β-derived peptide, which can be phosphorylated by the holoenzyme but not by the free catalytic subunits, we show that the holoenzyme activity requires the concurrent presence of both β and β′ encoding genes. Finally, conditions of nitrogen deprivation leading to a G0-like arrest result in a decrease of total CK2 activity, but have no effect on the activity of the holoenzyme. These findings newly indicate a regulatory role of β and β′ subunits of CK2 in the nutrient response.