Claudia Cirulli

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 (Peelman, F., Labeur, C., Vanloo, B., Roosbeek, S., Devaud, C., Duverger, N., Denefle, P., Rosier, M., Vandekerckhove, J., and Rosseneu, M. (2003) J. Mol. Biol. 325, 259-274). 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. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

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 interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.

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 β-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)2 as alkali reagent and (c) its further elimination by the simple addition of solid CO2 to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO3 by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine α-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures.

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). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective “precursor ion” and constant “neutral loss” triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.

The Ribosomal Protein L2 Interacts with the RNA Polymerase α 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. A clear description of the RNAPalpha protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPalpha. A tagged form of Escherichia coli RNAPalpha was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPalpha and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a beta-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase beta-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.

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.

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. Conversely, it has been also shown that metabolic intermediates may control HDACs activity. Consequently, the acetylation/deacetylation of metabolic enzymes and the ability of metabolic intermediates to modulate HDACs may represent a cross-talk connecting cell metabolism, transcription, and other HDACs-controlled processes in physiological and pathological conditions. CRITICAL ISSUES Since metabolic alterations and HDACs deregulation are important cancer hallmarks, disclosing connections among them may improve our understanding on cancer mechanisms and reveal novel therapeutic protocols against this disease. FUTURE DIRECTIONS High-throughput metabolic studies performed by using more sophisticated technologies applied to the available models of conditional deletion of HDACs in cell lines or in mice will fill the gap in the current understanding and open directions for future research.

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. The first section is focused on the development of new enrichment methods coupled to mass spectrometry. Thus, improved approach, based on simple chemical manipulations and mass spectrometric procedures, for the selective analysis of phosphoserine and phosphothreonine in protein mixtures, following conversion of the peptide phosphate moiety into DTT derivatives, is described. However the major aim of this work is devoted to the use of isotopically labelled DTT, thus allowing a simple and direct quantitative MS analysis. The final part of the work is focused on the development of a strategy to study phosphorylation without preliminary enrichment but using the high performance of a novel hybrid mass spectrometer linear ion trap.