Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family.
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PMID:Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase. 852 23

The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.
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PMID:Binding sites for adeno-associated virus Rep proteins within the human genome. 903 95

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol-chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15-20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15-20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC-CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.
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PMID:Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis. 918 82

A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.
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PMID:Antisense oligonucleotide of WAF1 gene prevents EGF-induced cell-cycle arrest in A431 cells. 948 44

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.
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PMID:Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111). 980 82

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.
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PMID:N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor. 1042 53

Endometrial glands contain higher levels of LH/hCG receptors than other cells in the human uterus. The present study investigated their functional importance. Northern and Western blotting and covalent receptor cross-linking demonstrated that human endometrial gland epithelial cells in culture contained multiple LH/hCG receptor transcripts and an 80-kDa receptor protein that can bind [125I]hCG in a hormone-specific manner. Culturing cells with highly purified hCG resulted in a time- and dose-dependent increase in steady state levels of cyclooxygenase-2 (COX-2) messenger ribonucleic acid and protein and the secretion of PGE2. Although human LH could mimic hCG, FSH, TSH, and alpha- or beta-subunits of hCG had no effect on COX-2 protein levels. Studies on signaling revealed that treatment of cells with hCG resulted in an increase in cAMP levels and protein kinase A (PKA) activity. Inhibition of PKA activity by cotreatment with isoquinoline-sulfonamide (H-89) prevented hCG from increasing COX-2 protein levels. Treatment with 8-bromo-cAMP mimicked the effect of hCG, and cotreatment with a selective inhibitor of type I PKA, 8-chloro-cAMP, prevented 8-bromo-cAMP and hCG from increasing COX-2 protein levels. The requirement of receptors for LH/hCG action was investigated by 24-h treatment of human endometrial gland epithelial cells with 21-mer phosphorothioate oligodeoxynucleotides (ODNs) synthesized from human receptor sequence. Treatment with 2 micromol/L antisense, but not sense, ODN resulted in a dramatic reduction in LH/hCG receptor protein levels. hCG was unable to increase COX-2 protein, PGE2, and cAMP levels in an antisense, but not in sense, ODN-treated cells. In summary, we conclude that hCG and LH treatment can increase expression of the COX-2 gene in human endometrial gland epithelial cells. The effect was time and dose dependent, hormone specific, and mediated by the cAMP/type I protein kinase A signaling pathway. The hCG actions require a normal complement of its receptors in cells. These hCG and LH effects may be another action of these hormones in human endometrium that is important for implantation of the blastocyst and continuation of pregnancy.
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PMID:Treatment of human endometrial gland epithelial cells with chorionic gonadotropin/luteinizing hormone increases the expression of the cyclooxygenase-2 gene. 1048 12

IkappaB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of IkappaBalpha, the regulatory subunit of the transcription factor NF-kappaB. A procedure for an IKK protein kinase assay is described that uses an in vivo biotinylated IkappaB protein substrate, [gamma-(33)P]ATP, and capture onto a streptavidin membrane. Residues 1-54 of the IkappaBalpha substrate were expressed as a fusion with glutathione S-transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacterial expression. Using the streptavidin capture assay the phosphorylation activities of recombinant IKK-1 and -2 were characterized. The assay provided a convenient way to compare IKK protein and peptide substrate preferences; biotinylated GST-IkappaBalpha(1-54) was more readily phosphorylated by both IKK-1 and IKK-2 compared to biotinylated myelin basic protein or a 20-mer biotinylated peptide containing serines 32 and 36 of IkappaBalpha. IKK-1 had 83-fold less activity than IKK-2, and the IKK-1+2 complex had approximately 2-fold more activity than IKK-2. IKK-1+2 and IKK-2 had similar K(m) values for ATP and GST-biotin-IkappaB(1-54) and were similarly inhibited by staurosporine and two of its analogues K252a and K252b, suggesting that most of the IkappaBalpha kinase activity in the IKK-1+2 complex may be attributed to IKK-2. Several features of the assay including the broad linear binding range of the streptavidin membranes for the protein substrate GST-biotin-IkappaB(1-54) (1-4000 pmol of protein/cm(2)), the low background, and its capacity for both biotinylated peptides and proteins make it a useful tool for quantitating IKK activity. These factors and the ease of expressing in vivo biotinylated GST fusions will make this assay approach suitable for a wide variety of protein kinases.
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PMID:Assay for IkappaB kinases using an in vivo biotinylated IkappaB protein substrate. 1052 19

We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call 'pseudo-cyclic oligonucleotides' (PCOs). PCOs contain two oligonucleotide segments attached through their 3'-3'- or 5'-5'-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5-8 nucleotides long and complementary to the 3'- or 5'-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RIalpha (PKA-RIalpha). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3'-exonuclease) or spleen (a 5'-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3'-3'- or 5'-5'-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.
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PMID:Pseudo-cyclic oligonucleotides: in vitro and in vivo properties. 1065 77

A novel 17-mer peptide ligand for cyclic AMP was designed using the amino acid sequences of essential subsites in various cyclic AMP-dependent protein kinase (protein kinase A) families. The Au disk electrode, which was modified with the designed 17-mer oligopeptide, responded to cyclic AMP but virtually did not respond to any other cyclic nucleotides using the ion channel sensor mechanism. On the other hand, a scrambled peptide, which had the same amino acid composition as and had an amino acid sequence different from the 17-mer oligopeptide, did not respond to any nucleotides. This indicates that the designed 17-mer peptide actually acted as a selective ligand for cyclic AMP. This ligand-designing strategy using peptide sequences in target-binding proteins may possibly be extended to the design of peptide ligands for other second messengers.
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PMID:The design of cyclic AMP--recognizing oligopeptides and evaluation of its capability for cyclic AMP recognition using an electrochemical system. 1102 28


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