Distal Recognition Sites in Substrates Are Required for Efficient Phosphorylation by the cAMP-Dependent Protein Kinase

Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.PROTEIN kinases are key mediators of signal transduction in all eukaryotic cells. Each protein kinase modifies a distinct set of substrates, and the biological consequences of activating any kinase are the result of the collective actions of these target proteins (Hunter 2000; Manning et al. 2002). The ability to identify substrates is therefore essential for a complete understanding of most signaling pathways. Unfortunately, this identification process tends to be difficult, and few physiologically relevant targets are known for most protein kinases (Manning and Cantley 2002; Johnson and Hunter 2005). This situation may be changing as a number of innovative approaches to this problem have been developed in recent years (reviewed in Ptacek and Snyder 2006; Deminoff and Herman 2007; Ubersax and Ferrell 2007).This article is focused on the cAMP-dependent protein kinase (PKA) from the budding yeast, Saccharomyces cerevisiae. The PKA enzyme is found in all eukaryotes and is one of the most intensely studied members of this protein family (Taylor et al. 2005). PKA was the first protein kinase structure to be described, and its structure has provided essential insights into the general organization and catalytic mechanism of these enzymes (Knighton et al. 1991; Smith et al. 1999). Subsequent work has illustrated the conserved nature of the protein kinase core and the different ways that the activity of these enzymes can be regulated (Hunter 2000; Huse and Kuriyan 2002; Kannan and Neuwald 2005). In S. cerevisiae, PKA activity is a key regulator of cell growth and the response to environmental stress (Toda et al. 1985; Thevelein and De Winde 1999; Herman 2002; Schneper et al. 2004). We are interested in understanding the role of PKA in these processes and have identified a number of substrates for this enzyme (Howard et al. 2003; Chang et al. 2004; Budovskaya et al. 2005; Deminoff et al. 2006). One of the approaches used for this identification took advantage of PKA variants that exhibit a stable binding to substrate proteins (Deminoff et al. 2006). This binding is novel as most wild-type protein kinases, including PKA, interact only transiently with their substrates (Manning and Cantley 2002). Interestingly, one of these PKA variants was altered at a residue that is conserved in all protein kinases, suggesting that it might be possible to generate substrate-binding versions of other enzymes in this family.These variants of PKA were used here to explore the nature of the protein kinase–substrate interaction. These studies identified substrate domains distal to the sites of phosphorylation that were required for efficient recognition by the wild-type PKA, both in vitro and in vivo. These substrate domains were found to interact with a C-terminal region of PKA that is itself removed from the active site of the enzyme. A systematic mutagenesis of PKA identified additional residues that, when altered, resulted in a stable association with substrates. These latter residues are in close proximity in the three-dimensional structure and may link the active site with this C-terminal substrate-binding domain of PKA. Finally, we show that similar alterations within a second protein kinase, the mammalian double-stranded RNA-dependent protein kinase (PKR), also led to an increased affinity for substrates. In all, the data suggest that the interactions described here may be generally important for protein kinase function and models that explain potential roles for these substrate domains are discussed.     

Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin

Transducin was [32P]ADP-ribosylated by cholera toxin in bovine retinal rod outer segments and then partially purified on omega-amino octyl agarose to remove other ADP-ribosylated proteins. Trypsin digestion of the ADP-ribosylated transducin and further purification using boronate-polyacrylamide beads and high performance liquid chromatography yielded a single radiolabeled tetrapeptide, Ser-Arg-Val-Lys. The ADP-ribose is linked to the guanidinium group of arginine.     

Natural Rabies Infection in a Domestic Fowl (Gallus domesticus): A Report from India

Background

Rabies is a fatal encephalitis caused by viruses belonging to the genus Lyssavirus of the family Rhabdoviridae. It is a viral disease primarily affecting mammals, though all warm blooded animals are susceptible. Experimental rabies virus infection in birds has been reported, but naturally occurring infection of birds has been documented very rarely.

Principal Findings

The carcass of a domestic fowl (Gallus domesticus), which had been bitten by a stray dog one month back, was brought to the rabies diagnostic laboratory. A necropsy was performed and the brain tissue obtained was subjected to laboratory tests for rabies. The brain tissue was positive for rabies viral antigens by fluorescent antibody test (FAT) confirming a diagnosis of rabies. Phylogenetic analysis based on nucleoprotein gene sequencing revealed that the rabies virus strain from the domestic fowl belonged to a distinct and relatively rare Indian subcontinent lineage.

Significance

This case of naturally acquired rabies infection in a bird species, Gallus domesticus, being reported for the first time in India, was identified from an area which has a significant stray dog population and is highly endemic for canine rabies. It indicates that spill over of infection even to an unusual host is possible in highly endemic areas. Lack of any clinical signs, and fewer opportunities for diagnostic laboratory testing of suspected rabies in birds, may be the reason for disease in these species being undiagnosed and probably under-reported. Butchering and handling of rabies virus- infected poultry may pose a potential exposure risk.     

Isorhamnetin Inhibits Proliferation and Invasion and Induces Apoptosis through the Modulation of Peroxisome Proliferator-activated Receptor γ Activation Pathway in Gastric Cancer

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3′-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.     

A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.     

Racial disparities in total ankle arthroplasty utilization and outcomes

IntroductionThe objective of this study was to examine the racial disparities in total ankle arthroplasty (TAA) utilization and outcomes.MethodsWe used the National Inpatient Sample (NIS) to study the time-trends. Race was categorized as White and Black. Utilization rates were calculated for the U.S. general population per 100,000. Hospital length of stay, discharge disposition and mortality after TAA were assessed. We used the Cochran Armitage trend test to assess time-trends from 1998 to 2011 and chi-square test to compare TAA utilization. We used analysis of variance or chi-squared test to compare the characteristics of Whites and Blacks undergoing TAA and logistic regression to compare mortality, length of stay and discharge to home versus medical facility.ResultsThe mean ages for Whites undergoing TAA were 62 years and for Blacks was 52 years. Significant racial disparities were noted in TAA utilization rates (/100,000) in 1998, 0.14 in Whites vs. 0.07 in Blacks (P < 0.0001; 2-fold) and in 2011, 1.17 in Whites vs. 0.33 in Blacks (P < 0.0001; 4-fold). Racial disparities in TAA utilization increased significantly from 1998 to 2011 (P < 0.0001). There was a trend towards statistical significance for the difference in the length of hospital stay in Blacks vs. Whites (52.9% vs. 44.3% with length of hospital stay higher than the median; P = 0.08). Differences in the proportion discharged to an inpatient medical facility after TAA, 16.6% Blacks vs. 13.4% Whites, were not significant (P = 0.36).ConclusionsThis study demonstrated significant racial disparities with lower TAA utilization and suboptimal outcomes in Blacks compared to Whites. Further studies are needed to understand the mediators of these disparities and to assess whether these mediators can be targeted to reduce racial disparities in TAA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0589-2) contains supplementary material, which is available to authorized users.     

Protein microarray signature of autoantibody biomarkers for the early detection of breast cancer

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.     

Effects of point substitutions on the structure of toxic Alzheimer's β-amyloid channels: atomic force microscopy and molecular dynamics simulations

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of β-amyloid (Aβ) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aβ peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aβ(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known β-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aβ from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted β-sheet-based channel structure.     

Zinc-Independent Folate Biosynthesis: Genetic,Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

GTP cyclohydrolase I (GCYH-I) is an essential Zn²⁺-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn²⁺-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn²⁺ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn²⁺-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn²⁺-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. ​(Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. ​(Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn²⁺ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH₄) pathway (Fig. ​(Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table ​Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).

TABLE 1.

Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteria^a