Andrea Venerando

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

Multiple phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38–64 of β-catenin are phosphorylated by CK1 on Ser-45 with low affinity (Km ≈1 mM), whereas intact β-catenin is phosphorylated at Ser-45 with very high affinity (Km ≈200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70–74 positions or a F74AA mutation in full-length β-catenin had no significant effect on CK1 phosphorylation efficiency. β-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131–181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in Km value. Implication of the first armadillo repeat in β-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of β-catenin from α-catenin, further improves CK1 phosphorylation efficiency, lowering the Km value to <50 nM, approximating the physiological concentration of β-catenin. In contrast, α-catenin, which interacts with the N-terminal region of β-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that β-catenin association with α-catenin and β-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

Modulation of mitochondrial K+ permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K(+) current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K(+) in the assay medium. The effects of p13 on ROS are mimicked by the K(+) ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K(+) vs. H(+) currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K(+) permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.

Identification of novel protein kinase CK1 delta (CK1δ) inhibitors through structure-based virtual screening

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimers disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).

Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.

Pyrvinium pamoate does not activate protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation

It has been reported that pyrvinium pamoate (PyrPam), an FDA (U.S. Food and Drug Administration)-approved anthelminthic drug, is a potent inhibitor of Wnt signalling by a mechanism which implies the direct activation of protein kinase CK1α. In the present paper, we provide data ruling out any direct stimulatory effect of PyrPam on CK1, by showing that the catalytic activity of CK1α and those of its isoforms δ and γ1 are not significantly affected by PyrPam when tested with the aid of specific peptide and protein substrates. Accordingly, cell treatment with PyrPam has no significant effect on the phosphorylation of β-catenin Ser(45). By contrast, the phosphorylation of β-catenin Thr(41) is increased upon cell treatment with PyrPam, through a mechanism that implies the upstream dephosphorylation of Akt/PKB (protein kinase B) and of GSK3 (glycogen synthase kinase 3). It can be concluded from the present study that PyrPam is not a bona fide activator of CK1, its perturbation of cell signalling pathways being mediated by a complex mechanism initiated by a fall in Akt phosphorylation whose down-regulation promotes reduced phosphorylation and activation of GSK3. Consistent with this, lysates of cells treated with PyrPam display enhanced protein phosphorylation which is unaffected by CK1 inhibition, while disappearing upon inhibition of GSK3. Our data are consistent with the observation that PyrPam ultimately inhibits Wnt signalling despite its lack of efficacy on CK1.

Phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) serine-511 by the combined action of tyrosine kinases and CK2: the implication of tyrosine-512 and phenylalanine-508

The cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s).

From phosphoproteins to phosphoproteomes: a historical account

The first phosphoprotein (casein) was discovered in 1883, yet the enzyme responsible for its phosphorylation was identified only 130 years later, in 2012. In the intervening time, especially in the last decades of the 1900s, it became evident that, far from being an oddity, phosphorylation affects the majority of eukaryotic proteins during their lifespan, and that this reaction is catalysed by the members of a large family of protein kinases, susceptible to a variety of stimuli controlling nearly every aspect of life and death. The aim of this review is to present a historical account of the main steps of this spectacular revolution, which transformed our conception of a biochemical reaction originally held as a sporadic curiosity into the master mechanism governing cell regulation, and, if it is perturbed, causing cell dysregulation.

CFTR mutations altering CFTR fragmentation

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTRs in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTRs fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Understanding protein kinase CK2 mis-regulation upon F508del CFTR expression

We review areas of overlap between nucleoside diphosphate kinase (NDPK; nm23) and two proteins manifesting an equivalent diversity of action, each with many thousands of publications. The first is a constitutively active protein kinase, CK2 (formerly casein kinase 2), that includes NDPK amongst its hundreds of targets. The second is an enigmatic member of the ATP-binding cassette (ABC) family of membrane pumps that normally hydrolyse ATP to transport substrates. Yet our unusual family member (ABCC7) is not a pump but, uniquely, acts as a regulated anion channel. ABCC7 is the cystic fibrosis transmembrane conductance regulator (CFTR), and we discuss the highly prevalent CFTR mutation (F508del CFTR) in terms of the uncertainties surrounding the molecular basis of cystic fibrosis that cloud approaches to corrective therapy. Using lysates from cells stably expressing either wild-type or F508del CFTR, incubated with the CK2 substrate GTP, we show that the phosphoproteome of F508del CFTR-expressing cells both differs from wild-type CFTR-expressing cells and is significantly enhanced in intensity by ∼1.5-fold (p < 0.05, paired t test with Bonferroni correction, n = 4). Phosphorylation is about 50% attenuated with a specific CK2 inhibitor. We propose that a new function may exist for the CFTR region that is commonly mutated, noting that its sequence (PGTIKENIIF508GVSYDEYRYR) is not only highly conserved within the C sub-family of ABC proteins but also a related sequence is found in NDPK. We conclude that a latent path may exist between mutation of this conserved sequence, CK2 hyperactivity and disease pathogenesis that might also explain the heterozygote advantage for the common F508del CFTR mutant .