The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2α belongs to the CMGC family of the eukaryotic protein kinases. CK2 is an important survival and stability factor in eukaryotic cells: its catalytic activity is elevated in a wide variety of tumors while its down-regulation can lead to apoptosis. Thus, CK2 is a valuable target for drug development and for chemical biology approaches of cell biological research, and small organic inhibitors addressing CK2 are of considerable interest. We describe here the complex structure between a C-terminal deletion mutant of human CK2α and the ATP-competitive inhibitor emodin (1,3,8-trihydroxy-6-methylanthraquinone, International Union of Pure and Applied Chemistry name: 1,3,8-trihydroxy-6-methylanthracene-9,10-dione) and compare it with a previously published complex structure of emodin and maize CK2α. With a resolution of 1.5 Å, the human CK2α/emodin structure has a much better resolution than its maize counterpart (2.6 Å). Even more important, in spite of a sequence identity of more than 77% between human and maize CK2α, the two structures deviate significantly in the orientation, in which emodin is trapped by the enzyme, and in the local conformations around the ligand binding site: maize CK2α shows its largest adaptations in the ATP-binding loop, whereas human CK2α shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite different in this respect.     

High phosphorylation of HBV core protein by two alpha-type CK2-activated cAMP-dependent protein kinases in vitro

Two alpha-type CK2-activated PKAs (CK2-aPKAIalpha and CK2-aPKAIIalpha) were biochemically characterized in vitro using GST-HBV core fusion protein (GST-Hcore) and GST-Hcore157B as phosphate acceptors. It was found that (i), in the absence of cAMP, these two CK2-aPKAs phosphorylated both Ser-170 and Ser-178 on GST-Hcore and Hcore157B; (ii) this phosphorylation was approx. 4-fold higher than their phosphorylation by cAMP-activated PKAs; and (iii) suramin effectively inhibited the phosphorylation of Hcore157B by CK2-aPKAIIalpha through its direct binding to Hcore157B in vitro. These results suggest that high phosphorylation of HBV-CP by two CK2-aPKAs, in the absence of cAMP, may be involved in the pregenomic RNA (pgRNA) encapsidation and DNA-replication in HBV-infected cells.     

The Protein Kinase CK2 Holoenzyme Structure Supports Proposed Models of Autoregulation and Trans-Autophosphorylation

Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2^Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2^Andante to form aggregates with completely blocked active sites may contribute to this phenotype.     

Eukaryotic initiation factor 2alpha kinase is a nitric oxide-responsive mercury sensor enzyme: potent inhibition of catalysis by the mercury cation and reversal by nitric oxide

The activity of one of the eukaryotic initiation factor 2alpha kinases, heme-regulated inhibitor (HRI), is modulated by heme binding. Here, we demonstrate for the first time that Hg2+ strongly inhibits the function of HRI (IC50=0.6 microM), and nitric oxide fully reverses this inhibition. Other divalent metal cations, such as Fe2+, Cu2+, Cd2+, Zn2+ and Pb2+, also significantly inhibit kinase activity with IC50 values of 1.9-8.5 microM. Notably, inhibition by cations other than Hg2+ is not reversed by nitric oxide. Our present data support dual roles of Hg2+ and nitric oxide in the regulation of protein synthesis during cell emergency states.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

Alterations in protein and gene expression within the barrel cortices of ZnT3 knockout mice: experience-independent and dependent changes

Much evidence exists for the involvement of vesicular zinc in neurotransmission and cortical plasticity. Recent studies have reported that mice deficient in zinc transporter-3 protein (ZnT3) and thus, vesicular zinc, have significant behavioural and biochemical deficits. Here, we examined whether phenotypic differences existed in the barrel cortices of ZnT3 KO mice using functional proteomics and quantitative PCR. Additionally, by manipulating whisker input, we also investigated experience-dependent changes in protein and gene expression, thereby assaying how cortical plasticity is different in the absence of vesicular zinc. The GABA metabolizing protein ABAT was observed in lower abundances consistently in KO mice. Several presynaptic proteins were identified that were abundant in differing amounts between the WT and KO groups in an experience-dependent manner. At baseline, we observed a decrease in the relative expression of Dlg4, Grin2a, Mt3, and Ntrkb genes in KO mice. The reduced expression of Nrtkb persisted with whisker plucking. These data demonstrate that fundamental changes in the expression of proteins and genes important in neurotransmission occur in the absence of vesicular zinc. Furthermore, the complement of experience-dependent changes were different between WT and KO mice, indicating that the lack of vesicular zinc affects the process of cortical plasticity.     

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage

Effective control of the Ca²⁺ homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca²⁺ concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca²⁺signaling at subcellular resolution. Members of the superfamily of EF-hand Ca²⁺-binding proteins are effective to either attenuate intracellular Ca²⁺ transients as stochiometric buffers or function as Ca²⁺ sensors whose conformational change upon Ca²⁺ binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca²⁺-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca²⁺-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca²⁺-binding proteins whose expression precedes that of many other Ca²⁺-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca²⁺ sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca²⁺-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca²⁺signaling under physiological and disease conditions in the nervous system and beyond.     

Cyclic AMP regulates the expression and nuclear translocation of RFC40 in MCF7 cells

We have previously shown that the regulatory subunit of PKA, RIalpha, functions as a nuclear transport protein for the second subunit of the replication factor C complex, RFC40, and that this transport appears to be crucial for cell cycle progression from G1 to S phase. In this study, we found that N(6)-monobutyryl cAMP significantly up-regulates the expression of RFC40 mRNA by 1.8-fold and its endogenous protein by 2.3-fold with a subsequent increase in the RIalpha-RFC40 complex formation by 3.2-fold. Additionally, the nuclear to cytoplasmic ratio of RFC40 increased by 26% followed by a parallel increase in the percentage of S phase cells by 33%. However, there was reduction in the percentage of G1 cells by 16% and G2/M cells by 43% with a concurrent accumulation of cells in S phase. Interestingly, the higher percentage of S phase cells did not correlate with a parallel increase in DNA replication. Moreover, although cAMP did not affect the expression of the other RFC subunits, there was a significant decrease in the RFC40-37 complex formation by 81.3%, substantiating the decrease in DNA replication rate. Taken together, these findings suggest that cAMP functions as an upstream modulator that regulates the expression and nuclear translocation of RFC40.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

The regulation of mRNA stability in mammalian cells: 2.0

Messenger RNA decay is an essential step in gene expression to set mRNA abundance in the cytoplasm. The binding of proteins and/or noncoding RNAs to specific recognition sequences or secondary structures within mRNAs dictates mRNA decay rates by recruiting specific enzyme complexes that perform the destruction processes. Often, the cell coordinates the degradation or stabilization of functional subsets of mRNAs encoding proteins collectively required for a biological process. As well, extrinsic or intrinsic stimuli activate signal transduction pathways that modify the mRNA decay machinery with consequent effects on decay rates and mRNA abundance. This review is an update to our 2001 Gene review on mRNA stability in mammalian cells, and we survey the enormous progress made over the past decade.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

Crosstalk of tight junction components with signaling pathways

Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.