Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fibronectin and Arg-Gly-Asp (RGD)- and/or Pro-His-Ser-Arg-Asn (PHSRN)-containing oligopeptides were immobilized onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signalling kinases in the adhesion of human primary blood-derived macrophages on these modified substrata was investigated. We demonstrated that the protein tyrosine kinase (PTK) or protein serine/threonine kinase (PSK) dependency and the PTK-PSK cross-talk compensation for macrophage adhesion varied dynamically with the substratum modification and the culture time. The inhibition of MAPK kinase (MAPKK) decreased macrophage adhesion on TCPS, whereas the inhibition of phosphoinositide-3 kinase (PI3 kinase) decreased macrophage adhesion on networks at 24 h. The PI3 kinase-protein kinase C (PKC)-MAPK cascade was involved in macrophage adhesion on fibronectin-preadsorbed TCPS or networks but not on fibronectin-grafted networks. This fibronectin-mediated adhesion signalling involved both RGD and PHSRN sequences in a form of G(3)RGDG(6)PHSRNG on TCPS but not on networks. Furthermore, G(3)RGDG(6)PHSRNG grafted onto networks evoked unique signalling in macrophage adhesion from that preadsorbed onto networks. Thus, macrophage adhesion and the role of selected signalling kinases were modulated by the substratum and the ligand conjugation method.
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PMID:Human macrophage adhesion on fibronectin: the role of substratum and intracellular signalling kinases. 1178 Nov 39

HNS-32 [N(1),N(1)-dimethyl-N(2)-(2-pyridylmethyl)-5-isopropyl-3,8-dimethylazulene-1- carboxamidine] (CAS Registry Number: 186086-10-2) is a newly synthesized azulene derivative. Computer simulation showed that its three dimensional structure is similar to that of the class Ib antiarrhythmic drugs, e.g., lidocaine or mexiletine. HNS-32 potently suppressed ventricular arrhythmias induced by ischemia due to coronary ligation and/or ischemia-reperfusion in dogs and rats. In the isolated dog and guinea pig cardiac tissues, HNS-32 had negative inotropic and chronotropic actions, prolonged atrial-His and His-ventricular conduction time and increased coronary blood flow. In the isolated guinea pig ventricular papillary muscle, HNS-32 decreased maximal rate of action potential upstroke (Vmax) and shortened action potential duration (APD). These findings suggest that HNS-32 inhibits inward Na+ and Ca2+ channel currents. In the isolated pig coronary and rabbit conduit arteries, HNS-32 inhibited both Ca2+ channel-dependent and -independent contractions induced by a wide variety of chemical stimuli. HNS-32 is a potent inhibitor of protein kinase C (PKC)-mediated constriction of cerebral arteries. It is likely to block both, Na+ and Ca2+ channels expressed in cardiac and vascular smooth muscles. These multiple ion channel blocking effects are largely responsible for the antiarrhythmic and vasorelaxant actions of HNS-32. This drug may represent a novel approach to the treatment of arrhythmias.
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PMID:A review of HNS-32: a novel azulene-1-carboxamidine derivative with multiple cardiovascular protective actions. 1183 Jul 49

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a basic 38-amino acid peptide, which acts through three main G protein-coupled VIP/PACAP receptor subtypes, called PAC1, VPAC1 and VPAC2. We have investigated the expression and function of PACAP and its receptors in the rat adrenal gland. Reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmune assay (RIA) allowed the detection of PACAP expression as mRNA and protein exclusively in adrenal medulla (AM). RT-PCR and quantitative autoradiography, using [(125)I]PACAP and selective VIP/PACAP receptor ligands, demonstrated the expression of PAC1 only in AM, and VPAC1 and VPAC2 in both AM and zona glomerulosa (ZG), PACAP receptor expression being absent in zona fasciculata/reticularis (ZF/R). PACAP38 concentration-dependently increased aldosterone secretion from dispersed ZG cells and catecholamine secretion from AM tissue, the maximal effective concentration being 10(-7) M. ZF/R cells did not display any secretory response to PACAP38. Aldosterone response of ZG cells to 10(-7) M PACAP38 was unaffected by the PAC1-antagonist (A) PACAP(6-38), and significantly decreased by the VPAC1-A [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP(3-7) GRF(8-27)-NH(2). Catecholamine response of AM tissue to PACAP38 was reduced, but not abolished, by both PAC1-A and VPAC1-A. The VPAC2 agonist (ago) Ro25-1553 elicited sizeable secretory responses from both ZG cells and AM tissue. PACAP38 (10(-7) M) evoked a marked rise in cyclic-AMP (cAMP) and inositol-1,4,5-triphosphate (IP3) production by ZG cells and AM tissue. cAMP response of ZG cells was lowered by VPAC1-A, and that of AM tissue by both PAC1-A and VPAC1-A. IP3 response of ZG cells and AM tissue was unaffected by PAC1-A and decreased by VPAC1-A. VPAC2-ago did not affect cAMP release, but raised IP3 production by both ZG cells and AM tissue. Aldosterone response of ZG cells and catecholamine response of AM tissue to PACAP38 (10(-7) M) were reduced by the adenylate cyclase (AC) and phospholipase-C (PLC) inhibitors (I) SQ-22536 and U-73122, as well as by the protein kinase (PK)A-I H-89 and PKC-I calphostin-C. Conversely, the secretory responses of both ZG and AM preparations to VPAC2-ago were annulled by PLC-I, lowered by PKC-I, and unaffected by either AC-I or PKA-I. Collectively, our findings allow us to conclude that in the rat adrenals: i) PACAP biosynthesis exclusively occurs in the AM; ii) ZG cells are provided with functional VPAC1 and VPAC2 receptors, whose activation by PACAP evokes a moderate aldosterone response; iii) AM cells possess all the subtypes of VIP/PACAP receptors, whose activation by PACAP elicits a marked catecholamine response; and iv) PAC1 receptors are coupled to the AC-dependent cascade, VPAC1 receptors to both the AC- and PLC-dependent cascades, and VPAC2 receptors exclusively to the PLC-dependent cascade.
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PMID:Pituitary adenylate cyclase-activating polypeptide and PACAP receptor expression and function in the rat adrenal gland. 1183 29

Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.
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PMID:Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay. 1194 69

Legumain (asparaginyl endopeptidase) was purified to homogeneity from bovine kidneys. The molecular mass of the purified enzyme was calculated to be 34000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol. The enzyme rapidly hydrolyzed the substrate Z-Ala-Ala-Asn-MCA and was strongly inhibited by N-ethylmaleimide, p-chloromercuribenzene-sulfonic acid, Hg(2+) and Cu(2+). The amino acid sequence of the first 26 residues of the enzyme was Gly-Gly-Lys-His-Trp-Val-Val-Ile-Val-Ala-Gly-Ser-Asn-Gly-Gln-Tyr-Asn-Tyr-Arg-His-Gln-Ala-Phe-Ala-Asp-His-. This sequence is highly homologous to the sequences in the N-terminal of pig kidney legumain. We screened a bovine kidney cortex cDNA library using a DNA probe that originated from rat legumain, and we determined the bovine kidney cDNA structure and deduced the amino acid sequence. The cDNA is composed 1934 bp and encodes 433 amino acids in the coding region. The enzyme was strongly stained in the proximal tubules of the rat kidney in an immunohistochemical study. Vitamin D-binding protein which is known to be a ligand to megalin existing in the proximal tubules, was cleaved in a limited proteolytic manner by bovine kidney legumain. These results suggested that legumain contributes to the processing of macromolecules absorbed by proximal tubule cells. The enzyme also cleaved an N-terminal synthetic peptide of bovine annexin II (Gly(24)-Ser-Val-Lys-Ala-Tyr-Thr(30)-Asn-Phe-Asp-Ala-Glu(35)-Arg-Asp(37)) at a position between Asn(31) and Phe(32). The amino-terminal domain of annexin II has p11 subunit binding sites and phosphorylation sites for both pp60(src) and protein kinase C. This suggests that legumain plays an important role in inactivation and degradation of annexin II, which is abundant in the receptor-recycling compartments of endosomes/lysosomes.
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PMID:Legumain from bovine kidney: its purification, molecular cloning, immunohistochemical localization and degradation of annexin II and vitamin D-binding protein. 1198 26

Two gap junction proteins, connexin43 (Cx43) and connexin45 (Cx45), are coexpressed in many cardiac and other cells. Homomeric channels formed by these proteins differ in unitary conductance, permeability, and regulation. We sought to determine the ability of Cx43 and Cx45 to oligomerize with each other to form heteromeric gap junction channels and to determine the functional and regulatory properties of these heteromeric channels. HeLa cells were transfected with Cx45 or (His)(6)-tagged Cx43 or sequentially transfected with both connexins. Immunoblots verified production of the transfected connexins, and immunofluorescence demonstrated that they were colocalized in the HeLa-Cx43(His)(6)/Cx45 cells. Connexons were solubilized from HeLa-Cx43(His)(6)/Cx45 cells by using Triton X-100 and were applied to a Ni(2+)-NTA column, which binds the His(6) sequence. Cx45 was coeluted from the column with Cx43(His)(6), demonstrating that some hemichannels contain both connexins. Single-channel recordings showed that the HeLa-Cx43(His)(6)/Cx45 cells exhibited single-channel conductances that were not observed in cells expressing either connexin alone. Dye-coupling experiments showed that HeLa-Cx43(His)(6) cells readily passed Lucifer yellow and N-(2-aminoethyl)biotinamide hydrochloride (neurobiotin); in contrast, HeLa-Cx45 and HeLa-Cx43(His)(6)/Cx45 cells showed extensive intercellular passage of neurobiotin but little coupling with Lucifer yellow. Treatment with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate reduced junctional conductance in cells expressing Cx43, Cx45, or both connexins, but it reduced the extent of neurobiotin transfer only in HeLa-Cx43(His)(6) and HeLa-Cx43(His)(6)/Cx45 cells but not in the HeLa-Cx45 cells. Thus, biochemical and electrophysiological evidence suggests that Cx43 and Cx45 extensively mix to form heteromeric channels; however, individual connexin components dominate aspects of the physiological behavior of these channels.
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PMID:Connexin43 and connexin45 form heteromeric gap junction channels in which individual components determine permeability and regulation. 1203

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.
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PMID:Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels. 1222 Oct 79

Calpain, a cytosolic cysteine protease, requires calcium ions for activity. It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C. More recently, calpain was shown to participate in apoptosis. In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain. 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively. 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV). 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively. 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His-. Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity. 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively. 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF). Eight clones were cancelled with wrong ORF. The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1. enzymes or proteins related to signal transduction in the cell; 2. contraction proteins 3. matrix proteins 4. unknown proteins. 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV. Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV. Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait. Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located. Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.
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PMID:Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system. 1235 55

Glutamate transporters play an important role in homeostasis of extracellular glutamate, a major excitatory neurotransmitter and a potential neurotoxin. In mammalian brain, glutamate transporter type 2 (EAAT2) is the most abundant form. Studies of molecular structures demonstrated that tyrosine 403 is critical in regulating the ion selectivity and transport mode of EAAT2. We hypothesized that wild type EAAT2 and its mutant at tyrosine 403 have different responses to volatile anesthetics, commonly used anesthetics that have been shown to affect glutamate transporter activity and decrease extracellular glutamate concentrations. We used site-directed mutagenesis and oocyte expression systems to test the hypothesis. Volatile anesthetics did not affect the activity of wild type EAAT2, isolated from rat hippocampus. When tyrosine 403 was replaced by histidine (Y403H), volatile anesthetics (isoflurane or halothane) at clinically relevant concentrations significantly decreased the transporter activity. Okadaic acid, a phosphatase inhibitor, significantly prolonged the isoflurane-induced inhibition. This inhibition was reversed by staurosporine and calphostin C, two protein kinase C (PKC) inhibitors, but not by the third PKC inhibitor, chelerythrine. Phorbol 12-myristate 13-acetate, a PKC activator, inhibited the activity of both wild type and Y403H EAAT2. This inhibition was also reversed by the same two PKC inhibitors but not by the third one. These results suggest that the switch of tyrosine 403 to histidine rendered EAAT2 sensitive to volatile anesthetics, a phenomenon that may require protein phosphorylation. PKC may be involved in the regulation of the activity of both wild type and Y403H EAAT2.
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PMID:The different responses of rat glutamate transporter type 2 and its mutant (tyrosine 403 to histidine) activity to volatile anesthetics and activation of protein kinase C. 1238 59

We showed recently that human O6-alkylguanine-DNA alkyltransferase (AGT), a key target for enhancing the efficacy of anticancer alkylating agents, is regulated by phosphorylation in brain tumor cells. This report describes the problems we encountered in using a glutathione S-transferase (GST)-tagged AGT as the substrate in our search for cellular AGT kinases, validation of a new pull-down assay for AGT phosphorylation, and its wide applicability for quantitating protein kinases in crude extracts and purified fractions. The GST-tag present in the fusion protein, by itself, was found to undergo significant phosphorylation by tumor cell extracts and contribute to spurious results. Instead, we used a histidine-tagged AGT protein, and its micro-scale purification with Talon resin as the basis for a quantitative pull-down assay, and applied it for measuring AGT phosphorylation by protein kinase C (PKC) and other cellular kinases. The pull-down procedure can be easily adopted for quantitating protein kinases in a variety of settings, as it overcomes the need for substrate immunoprecipitation when whole cell extracts are used, and eliminates the autophosphorylated kinase proteins, when purified kinases are used. Our observations call for caution in interpreting the results with GST-fusion proteins in phosphorylation studies.
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PMID:Phosphorylation of O6-alkylguanine-DNA alkyltransferase: experience with a GST-fusion protein and a new pull-down assay. 1243 Jan 83


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