Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness.

New Insights into the Connection Between Histone Deacetylases, Cell Metabolism, and Cancer.

Significance: Histone deacetylases (HDACs) activity and cell metabolism are considered important targets for cancer therapy, as both are deregulated and associated with the onset and maintenance of tumors.

Recent Advances: Besides the classical function of HDACs as HDAC enzymes controlling the transcription, it is becoming increasingly evident that these proteins are involved in the regulation of several other cellular processes by their ability to deacetylate hundreds of proteins with different functions in both the cytoplasm and the nucleus. Importantly, recent high-throughput studies have identified as important target proteins several enzymes involved in different metabolic pathways.

Protein kinase CK2 holoenzyme promotes start-specific transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the entrance into S phase requires the activation of a specific burst of transcription, which depends on SBF (SCB binding factor, Swi4/Swi6) and MBF (MCB binding factor, Mbp1/Swi6) complexes. CK2 is a pleiotropic kinase involved in several cellular processes, including the regulation of the cell cycle. CK2 is composed of two catalytic subunits (α and α') and two regulatory subunits (β and β'), both of which are required to form the active holoenzyme.

Mitochondrial complex I inhibitors and forced oxidative phosphorylation synergize in inducing cancer cell death.

Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells.

A surface-activated chemical ionization approach allows quantitative phosphorylation analysis of the cyclin-dependent kinase inhibitor Sic1 phosphorylated on Ser201.

Rationale: Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations.

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 K(m) for both the ATP and the peptide substrate RRRADDSDDDDD, while a significant increase in V (max) is observed.

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.

Proteomics and systems biology to tackle biological complexity: Yeast as a case study.

In this note we discuss how, by using budding yeast as model organism (as has been done in the past for biochemical, genetics and genomic studies), the integration of "omics" sciences and more specifically of proteomics with systems biology offers a very profitable approach to elucidating regulatory circuits of complex biological functions.

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.

The ribosomal protein L2 interacts with the RNA polymerase alpha subunit and acts as a transcription modulator in Escherichia coli.

Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP alpha subunit (RNAPalpha) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process.

The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: new hints for actin folding.

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner.

The analysis of phosphoproteomes by selective labelling and advanced mass spectrometric techniques.

This chapter focuses on the development of new proteomic approaches based on classical biochemical procedures coupled with new mass spectrometry methods to study the phosphorylation, the most important and abundant PTMs in modulating protein activity and propagating signals within cellular pathways and networks. These phosphoproteome studies aim at comprehensive analysis of protein phosphorylation by identification of the phosphoproteins, exact localization of phosphorylated residues, and preferably quantification of the phosphorylation. Because of low stoichiometry, heterogeneity, and low abundance, enrichment of phosphopeptides is an important step of this analysis.

cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins.

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3'-UTR.

Identification of free phosphopeptides in different biological fluids by a mass spectrometry approach.

Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring).

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 cka1Deltacka2-8(ts) strain.

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature.

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic alpha-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins.

Membrane proteome in Escherichia coli probed by MS3 mass spectrometry: a preliminary report.

Characterization of the membrane proteome is particularly intriguing since a better knowledge in this field might lead to new insights into the function of different membrane systems. Despite the biological relevance of surface proteins however, their characterization still remains a challenging task. Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment.

Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation.

Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification.

Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2.

Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry.

Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified the beta-elimination reaction to get the corresponding alkene moiety from the phosphate esters.