20,949 research outputs found
Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution
No full textUdgivelsesdato: 1998-May-1CK2alpha is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 A resolution, of recombinant maize CK2alpha (rmCK2alpha) in the presence of ATP and Mg2+, shows the enzyme in an active conformation stabilized by interactions of the N-terminal region with the activation segment and with a cluster of basic residues known as the substrate recognition site. The close interaction between the N-terminal region and the activation segment is unique among known protein kinase structures and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP and GTP as phosphorylating agents
Mutational analysis of residues implicated in the interaction between protein kinase CK2 and peptide substrates
No full textSixteen derivatives of the optimal peptide substrate RRRA-DDSDDDDD in which aspartic acids were singly or multiply substituted by alanine have been assayed for their phosphorylation efficiency by either wild type protein kinase CK2 or CK2 alpha mutants defective in substrate recognition. With wild type CK2, the only detrimental single substitutions were those at positions +3 and +1. Each of these caused a 5-fold increase of Km and a 2-fold decrease of the Vmax values. If both aspartic acids at n + 1 and n + 3 were substituted however, the Km rose 24-fold and the Vmax decreased 16-fold. Multiple substitutions tend to have a more than additive effect even if they affect individually dispensable aspartic acids; thus, double, triple, and quintuple substitutions at positions n - 2 and -1, and n + 2, +4, and +5 had detrimental consequences comparable to those observed with substitutions at n + 1 and n + 3. These data indicate that additional acidic residues besides those at n + 1 and n + 3 are collectively required for efficient phosphorylation of CK2 substrates. They are also consistent with a flexible mode of binding of the substrate, where acidic residues may play interchangeable roles. Among twelve CK2 mutants in which basic residues suspected to be implicated in substrate recognition have been replaced by alanine, only K74-77A, K79R80K83A, R191,195K198A, and K198A showed substantially increased Km values with the optimal substrate RRRA-DDSDDDDD, symptomatic of a reduced ability to bind it. However, if the suboptimal substrate RRRA-AASDDDDD was used, the single mutants K49A, K71A, K77A, R80A, and H160A also exhibited Km values significantly higher than those of wild type CK2. Kinetic analysis with singly substituted derivatives of peptide RRRA-DDSDDDDD revealed that K49 is implicated in the recognition of the determinant at position n + 2, K77 cooperates with other residues nearby in the interaction with the determinants at n + 3 and n + 4, while K198 plays a prominent role in the recognition of the determinant at n + 1
CK2: a protein kinase in need of control
No full textProtein kinase CK2 is a heterotetrameric alpha2beta2 Ser/Thr protein kinase with some features unusual among the eukaryotic protein kinases: (1) CK2 recognizes phosphoacceptor sites specified by several acidic determinants; (2) CK2 can use both ATP and GTP as phosphoryl donors; and (3) the regulatory properties of CK2 are poorly understood; it is insensitive to any known second messenger and displays high basal activity. To gain insight into CK2 regulation and to understand its unusual properties, site-directed mutagenesis experiments on both subunits and X-ray crystallographic studies of the catalytic alpha-subunit were performed. The noncatalytic beta-subunit has at least three functions: (1) it protects the alpha-subunit against denaturing agents or conditions; (2) it alters the substrate specificity of the alpha-subunit; and (3) it modulates the activity of the enzyme, i.e., depending on the substrate, it increases or decreases the activity of the alpha-subunit. Mutagenesis experiments revealed that an acidic stretch between amino acids 55 and 64 has a down-regulatory and autoinhibitory function. Mutational analysis of the alpha-subunit has revealed a network of unique basic residues that are responsible for the recognition of phosphoacceptor substrates and for down-regulation by the beta-subunit and by polyanionic inhibitors. The resolution of the crystal structure of Zea mays CK2 alpha-subunit has disclosed the structural features that are responsible for high basal activity and for unusual response to nucleotide analogs. The increasing knowledge of CK2 structure-function relationships will allow the design of highly selective inhibitors of this pleiotropic kinase with oncogenic potential
CK2 Function in the Regulation of Akt PathwayProtein Kinase CK2 Cellular Function in Normal and Disease States
No full textThe crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.
No full textThe crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate
2-Triazenoazaindoles: Á novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells
No full textPancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinase
Turbocharger speed monitoring based on vibration measurements for diesel engine management
No full textThe Ste locus, a component of the selfish cry-Ste system of Drosophila melanogaster, encodes a protein mimicking some properties of the beta subunit of casein kinase 2.
No full textMales of Drosophila melanogaster lacking the
Y chromosome-linked crystal locus show multiple meiotic
alterations, including chromosome disorganization and
prominent crystal formation in primary spermatocytes. These
alterations are due to the derepression of the X chromosomelinked
Stellate sequences. To understand how the derepression
of the Stellate elements gives rise to these abnormalities,
we have expressed the protein encoded by the Stellate sequences
in bacteria and produced an antibody against the
fusion protein. Immunostaining of crystal- testes has clearly
shown that the Stellate protein is a major component of the
crystals. Moreover, in vitro experiments have shown that this
protein can interact with the catalytic a subunit of casein
kinase 2 enzyme, altering its activity
A Systematic Review of Industry 4.0 Maturity Models: Applicability in the O&G Upstream Industry
No full textThe study aims to review the currently available Industry 4.0 (I4.0) maturity models (MMs) for manufacturing industries and analyse their applicability in the oil and gas (O&G) upstream sector. Knowing that the growth in demand for energy through crude oil and natural gas is still viable over the next decade, there is the drive to ensure sustenance and improvement in production. The study sees an opportunity in harnessing the gains of Industry 4.0 technologies for better solution-driven strategies in production processes, equipment availability and reliability which would translate into higher production performance. So, a review on the Industry 4.0 MMs is considered important. A systematic and in-depth literature review was performed to identify the specific requirements of this industry. This study examined the key characteristics of the O&G upstream sector and identified research gaps that need to be addressed to successfully support this industry for Industry 4.0 implementation. An Industry 4.0 MM that reflects the industrial realities for this industry more accurately from insights drawn from reviews of existing MMs is proposed. The review of 19 selected Industry 4.0 MMs revealed that the existing MMs are not a direct fit for the O&G upstream industry. Only a few of the models were clear on validation but with subjectivity, low number of persons and industries involved as limitations; none of the models confirmed validation with the O&G industry. There are varying views on the model dimensions and maturity levels by each author and not all required areas specific to the O&G industries were acknowledged by the models. An MM specific to this industry is therefore required. Although the journey of digitisation has commenced in the O&G industry, a reduction with the challenges of transition towards Industry 4.0 implementation and provision of support for improved efficiency is assured using a robust MM, as proposed in this paper
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