DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3-OH capping group from the DNA products generated by incorporating the 3-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.sequencing by synthesis ͉ DNA chip T he completion of the Human Genome Project (1) was a monumental achievement in biological science. The engine behind this project was the Sanger sequencing method (2), which is still the gold standard in genome research. The prolonged success of the Sanger sequencing method is because of its efficiency and fidelity in producing dideoxy-terminated DNA products that can be separated electrophoretically and detected by fluorescence (3-5). However, a challenge in the use of electrophoresis for DNA separation is the difficulty in achieving high throughput and the complexity involved in the automation, although some level of increased parallelization may be achieved by using miniaturization (6).To overcome the limitations of the Sanger sequencing technology, a variety of new methods have been investigated. Such approaches include sequencing by hybridization (7), mass spectrometry sequencing (8, 9), sequencing by nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (12), sequencing of single DNA molecules (13,14), and polymerase colonies (15) have been widely explored. Previously, we reported the development of a general strategy to rationally design cleavable fluorescent nucleotide reversible terminators (NRTs) ...
Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC␦ actions in cardiomyocytes. Rather, tyrosine phosphorylation regulates PKC␦ kinase activity. PKC␦ is recovered from the soluble fraction of H 2 O 2 -treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC␦ phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC␦ substrate specificity. The stimulus-specific modes for PKC␦ signaling identified in this study allow for distinct PKC␦-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes. Protein kinase C (PKC)1 comprises a multigene family of at least 10 structurally distinct phospholipid-dependent serinethreonine kinases that regulate cardiac contraction, play a role in ischemic preconditioning, and contribute to the pathogenesis of cardiac hypertrophy and heart failure (1, 2). PKC isoforms are single polypeptide chains with structurally homologous C-terminal catalytic domains and more variable N-terminal regulatory domains. This diverse group of enzymes is subdivided into three distinct subfamilies based upon structural differences in their N-terminal regulatory domain that confer distinct patterns of cofactor activation. Conventional PKC isoforms (cPKCs; ␣, I, II, ␥) contain an autoinhibitory pseudosubstrate domain followed by membrane-targeting C1 and C2 domains that are regulated by diacylglycerol (DAG) and calcium, respectively. Novel PKCs (nPKCs; ␦, ⑀, , and ) lack a calcium-binding C2 domain and are maximally activated by DAG and phorbol ester, in the absence of calcium. Atypical PKCs (aPKCs; and /) are regulated by lipids, but are not activated by second messengers such as calcium and DAG. Current models of PKC isoform activation in the heart have focused largely on the conformational changes induced by cofactor interactions with N-terminal membrane-targeting modules that anchor the enzyme to membranes, expel the autoinhibitory pseudosubstrate domain from the substrate-binding pocket, and thereby relieve autoinhibition. According to this model, individual PKC isoforms elicit distinct (and occasionally functionally opposing) cellular responses as a result of cofactorinduced compartmentation to distinct membrane subdomains, in close proximity to their unique sets of target protein substrates (1).Recent studies identify an additional mechanism for PKC regulation via sequential phosphorylations on a conserved threonine in the activation loop and two conserved serine/threonines in turn and hydrophobic motifs in the C terminus (3). For cPKCs, these phosphorylation even...
Abstract-Proteases elaborated by inflammatory cells in the heart would be expected to drive cardiac fibroblasts to proliferate, but protease-activated receptor (PAR) function in cardiac fibroblasts has never been considered. This study demonstrates that PAR-1 is the only known PAR family member functionally expressed by cardiac fibroblasts and that PAR-1 activation by thrombin leads to increased DNA synthesis in cardiac fibroblasts. The increase in DNA synthesis induced by PAR-1 substantially exceeds the effects of other G protein-coupled receptor agonists in this cell type. PAR-1 stimulates phosphoinositide hydrolysis and mobilizes intracellular calcium via pertussis toxin (PTX)-sensitive and PTX-insensitive pathways. Activation of PAR-1 leads to an increase in Src, Fyn, and epidermal growth factor receptor (EGFR) phosphorylation, with EGFR receptor transactivation by Src family kinases the major mechanism for PAR-1-dependent activation of extracellular signal-regulated kinase, p38-mitogen-activated protein kinase, and protein kinase B. Activation of PAR-1 also leads to an increase in DNA synthesis. PAR-1 signaling is highly contextual in nature, inasmuch as PAR-1 activates extracellular signal-regulated kinase and only weakly stimulates protein kinase B via a pathway that does not involve EGFR transactivation in cardiomyocytes. PAR-1 responses in cardiac fibroblasts and cardiomyocytes are predicted to contribute importantly to remodeling during cardiac injury and/or inflammation. Key Words: thrombin Ⅲ protease-activated receptors Ⅲ cardiac fibroblasts Ⅲ epidermal growth factor receptors Ⅲ signal transduction C ardiomyocytes occupy as much as 75% of cardiac mass but constitute only about one third of the total cell number in the heart. The remaining noncardiomyocytes consist mainly of interstitial cardiac fibroblasts, which provide structural support for cardiomyocytes, regulate extracellular matrix, and are the source of paracrine growth factors. Fibroblast proliferation and synthesis of matrix is essential for scar formation at sites of myocardial infarction. However, replacement of necrotic myofibrils by noncontractile fibrotic scar tissue disrupts the transmission of electrical impulses. Extensive fibroblast-induced adverse structural remodeling also occurs at distal noninfarcted segments of the ventricle, where it leads to diastolic stiffness and ultimately contributes to mechanical failure.Cardiac fibroblast growth and fibrillar collagen synthesis is highly regulated by humoral and mechanical stimuli. Angiotensin II (Ang II) and endothelin have been implicated as important mediators of interstitial remodeling in the context of hypertension, coronary heart disease, myocarditis, and congestive heart failure. 1 Ang II acts via a G protein-coupled receptor (GPCR) to stimulate a spectrum of biochemical signals that culminate in the expression of growth-associated nuclear protooncogenes and mitogenesis. Initial studies implicated G i protein ␥ subunits, Src family tyrosine kinases, tyrosine phosphorylation of ...
Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser 744 Protein kinase D1 (PKD1)2 is the founding member of a family of three related serine/threonine kinases that share a similar structural architecture and control a large number of biological processes that influence cell growth, differentiation, migration, and apoptosis (1, 2). PKDs have an N-terminal regulatory domain containing tandem cysteine-rich C1A/C1B domains that bind diacylglycerol-/phorbol ester-enriched membranes with high affinity and a pleckstrin homology (PH) domain that participates in protein-protein interactions. PH domain-dependent autoinhibitory intramolecular interactions maintain the enzyme in an inactive state, with low basal activity, in resting cells. PKD isoforms are activated by agonists that promote diacylglycerol accumulation and activate novel PKC (nPKC) isoforms at membranes. nPKCs activate PKD isoforms by phosphorylating a pair of highly conserved serine residues (Ser 744 /Ser 748 in PKD1, nomenclature based upon rodent sequence) in the kinase domain activation loop. This post-translational modification relieves autoinhibition and stabilizes the activation loop in a conformation that is optimized for catalysis. PKD1 then undergoes a series of autophosphorylation reactions at a cluster of serine residues at Ser 205 /Ser 208 and Ser 219 /Ser 223 in the regulatory C1A/C1B interdomain region and at Ser 916 at the extreme C terminus. These autophosphorylation reactions create docking sites for PKD1 binding partners and influence PKD1 localization within the cell (3, 4). A recent study also identified PKD1 autophosphorylation at the activation loop (primarily at Ser 748 ) during the chronic phase of PKD1 activation in bombesin-treated COS-7 cells (5); the relative roles of autocatalytic versus PKC-dependent activation loop phosphorylation in other cellular contexts has never been considered.PKD has emerged as a physiologically important signaling enzyme in many cell types. However, the list of known PKD substrates remains relatively short. We and others recently implicated PKD as a CREB-Ser 133 kinase that regulates Cre-dependent transcriptional responses (6, 7). PKD also functions as a physiologically relevant HDAC5 kinase (8). HDAC5 is a signal-responsive repressor of pathological cardiac remodeling (9, 10). PKD neutralizes the antihypertrophic actions of HDAC5, leading to the activation of a pathologic gene program that culminates in cardiac hypertrophy and ventricular remodeling. PKD also phosphorylates cardiac troponin I (cTnI), the "inhibitory" subunit of the troponin complex that "fine-tunes" myofilament function to hemodynamic load. cTnI contains three * This work was supported, in whole or in part, by National Institutes of Health Grant HL 77860 (USPHS NHLBI). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adve...
p53 controls the cellular response to genotoxic stress through multiple mechanisms. We report here that p53 regulates DUSP1, a dual-specific threonine and tyrosine phosphatase with stringent substrate specificity for mitogen-activated protein kinase (MAPK). DUSP1 is a potent inhibitor of MAPK activity through dephosphorylation of MAPK. In a colon cancer cell line containing inducible ectopic p53, DUSP1 protein level is significantly increased upon activation of p53, leading to cell death in response to nutritional stress. In mouse embryo fibroblast cells, DUSP1 protein abundance is greatly increased after oxidative stress in a p53-dependent manner and also when apoptosis is triggered. We show that p53 induces the activity of a human DUSP1 regulatory region. Furthermore, p53 can physically interact with the DUSP1 regulatory region in vivo, and p53 binds to a 10-bp perfect palindromic site in this DUSP1 regulatory region. We show that overexpression of DUSP1 or inhibition of MAPK activity significantly increases cellular susceptibility to oxidative damage. These findings indicate that p53 is a transcriptional regulator of DUSP1 in stress responses. Our results reveal a mechanism whereby p53 selectively regulates target genes and suggest a way in which subgroups of those target genes might be controlled independently. (Mol Cancer Res 2008;6(4):624 -33)
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