Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin. 802 8

The protein kinase inhibitors (PKIs) are potent inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase. In this study, the interaction between Phe10 of PKI and the C subunit residues Tyr235 and Phe239 was investigated using site-directed mutagenesis. Previous peptide studies as well as the crystal structure suggested that these residues may play a key role in C-PKI binding. The C subunit codons for Tyr235 and Phe239 were changed singly and in combination to serine codons. The mutated C alpha proteins were overexpressed in Escherichia coli. The purified C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins did not exhibit any differences in their Km(app) for the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) or Vmax(app), with respect to wild-type C alpha. All of the C subunit mutants displayed less than 2-fold changes in their Km(app) for ATP. The PKI alpha isoform displayed increased IC50 values for C alpha Y235S (71-fold), C alpha F239S (150-fold), and C alpha Y235S/F239S (1800-fold). Similarly, the PKI beta 1 protein showed increased IC50 values against the C alpha Y235S, C alpha F239S, and C alpha Y235S/F239S proteins, 9.4-, 11-, and 44-fold, respectively. In addition, the PKI alpha F10 codon was altered to an alanine codon, and this mutation decreased its ability to inhibit C alpha kinase activity, but did not affect its ability to inhibit C alpha Y235S/F239S. The mutation of Tyr235 and Phe239 to serines, however, did not alter the ability of the type II R subunit to inhibit phosphotransferase activity. These results suggest that C alpha Y235 and C alpha F239 are important for specific inhibition by both PKI alpha and PKI beta but not the type II R subunit and that mutations at these residues would be useful for in vivo analysis of C-PKI interactions.
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PMID:Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors. 802 74

The activation of a phosphatidylinositol (PI) 3-kinase is one of the earliest intracellular responses to T lymphocyte stimulation. We have used a human T cell line, Jurkat, and an antibody that recognizes the clonotype of its T cell receptor (TcR) to characterize the association of PI 3-kinase with the TcR and its activation in response to TcR cross-linking. We show that the TcR is constitutively associated with the alpha isoform of the PI 3-kinase p85 regulatory subunit. Stimulation of Jurkat cells through the TcR results in the rapid and transient activation of the associated PI 3-kinase. In addition, our results indicate that the tyrosine kinase pp56lck is essential for PI 3-kinase activation via the TcR.
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PMID:T cell receptor-associated alpha-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck. 803 11

The activity and expression of Ca(2+)-dependent cPKC alpha and beta isoenzymes in the particulate, soluble (cytosolic) and nuclear fractions of rat liver and the expression of Ca(2+)-independent nPKC delta and aPKC zeta were examined during the early stages (30 and 60 min, 24 and 96 h and 7 and 60 days post-hepatectomy) of the Solt-Farber 'resistant hepatocyte' model of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in Fischer 344 rats and related to the presence of gamma-glutamyl transpeptidase (GGT)-positive hyperplastic cell foci and persistent nodules in rat liver. Total PKC activity was unmodified by the carcinogenic treatment. In contrast, the PKC activity in the particulate, as well as nuclear fractions increased with time, reaching a maximum 60 days post-hepatectomy, with a decrease in the cytosolic activity. In carcinogen-treated animals maximal expression of cPKC alpha and beta isoenzymes was present 7 days post-hepatectomy, while no changes in nPKC delta and aPKC zeta immunoreactivity were detected. In the nucleus, no cPKC alpha isoform expression was observed, the cPKC beta expression being maximal at 60 days. Seven and 60 days post-hepatectomy GGT-positive hyperplastic cell foci and persistent nodules were present in rat liver respectively. Taken together, the results of this study suggest a role for nuclear cPKC beta and for cPKC alpha in promoting the selective growth of carcinogen-initiated hepatocytes in rat liver. No evidence for a role of Ca(2+)-independent nPKC delta and aPKC zeta isoenzymes in the early stages of DENA-induced liver carcinogenesis could be demonstrated.
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PMID:Membrane and nuclear protein kinase C activation in the early stages of diethylnitrosamine-induced rat hepatocarcinogenesis. 805 57

To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 microM. DNA synthesis, however, was already significantly inhibited at 0.05 microM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [3H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 microM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo II beta isoform is involved in the very early phases of lymphocyte activation, with function of the Topo II alpha isoform, which is more sensitive to VM26, being required for progression through S phase.
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PMID:Role of topoisomerase II in the structural and functional evolution of mitogen-stimulated lymphocyte nuclei. 808 36

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37,000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37,000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominantly the alpha isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co2+ or Mn2+ alone but not by Ca2+, Mg2+, Ni2+, Cu2+ or Zn2+. Activation of the enzyme was not reversed by removal of Co2+, but Mn(2+)-activated phosphatase activity was partially reversed when Mn2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co2+. Since phosphatase 1 alpha is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.
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PMID:A latent form of protein phosphatase 1 alpha associated with bovine heart myofibrils. 808 38

We performed an immunohistochemical study on rat brain and liver during fetal and neonatal life using rabbit antipeptide polyclonal antibodies able to recognize each thyroid hormone receptor (TR) isoform. The expression of TR alpha-1, alpha-2 and beta-1 proteins from 14 days of gestation to 21 days after birth was evaluated. Frozen tissues from 14 (F14), 17 (F17) and 21 (F21)-day-old fetuses and from 5 (N5), 16 (N16) and 21 (N21)-day old newborn rats were stained with anti-TR antibodies using an avidin-biotin-peroxidase system. The antipeptide antibodies utilized in the present study were characterized previously: alpha-144 antibody recognizes both TR alpha-1 and alpha-2; alpha-2-431 antibody is specific for TR variant alpha-2, and beta-62 antibody specifically reacts with the TR beta-1 isoform. The expression of TR alpha-1 was deduced by comparing the staining obtained with alpha-144 and alpha-2-431 antibodies. We demonstrated that each TR isoform is expressed in rat brain from 14 days of gestation and that the alpha isoform was predominant in the early stage. The three TR isoforms were expressed in both neural cell nuclei and in glial cell nuclei. As far as the liver is concerned, at F14 the expression of TR isoforms was weaker in hepatocytes when, on the contrary, TR alpha was clearly detected in hematopoietic cells. The expression of TRs in hepatocytes becomes evident later. The data that we obtained, although not quantitative, emphasize the presence of each TR isoform in brain and liver from 14 days of fetal rat life.
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PMID:Evaluation of the ontogeny of thyroid hormone receptor isotypes in rat brain and liver using an immunohistochemical technique. 812 84

Transactivation of the firefly luciferase-encoding vectors, pXP1 and pXP2, by the alpha isoform of CCAAT/enhancer-binding protein (C/EBP) is reported. Thus, these vectors are not 'promoterless' in every cellular context, and transactivation by C/EBP or a closely related factor should be considered as a possible explanation for the relatively high background levels of luciferase production that are occasionally observed after transfection of certain cells with these or other similar vectors.
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PMID:Transactivation of the 'promoterless' luciferase-encoding vectors pXP1 and pXP2 by C/EBP alpha. 812 11

Basal and stimulated adenylyl cyclase activities and Gs and Gi protein alpha-subunit levels (Gs alpha and Gi alpha) were compared in postmortem frontal cortex from 18 suicide cases and 22 matched controls. Basal, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated and forskolin stimulated enzyme activities were significantly lower in the suicide cases, compared to controls. These effects were most apparent in those suicides that had died from violent means or that had had a history of depression and appeared to reflect the lowered basal activity rather than a reduced ability of either GTP gamma S or forskolin to activate the enzyme. No significant correlations were found between adenylyl cyclase activity and either subject age or postmortem delay. Western blotting revealed no significant differences in Gs alpha and Gi alpha levels between control and suicide cases. However, levels of the smaller Gs alpha isoform (Gs alpha-S) showed a tendency to be increased in the violent death suicide and depressed suicide subgroups, compared to controls. Levels of the larger Gs alpha isoform (Gs alpha-L) showed a significant positive correlation with subject age. Gi alpha levels showed a significant negative correlation with subject age and a positive correlation with postmortem delay. These results support the hypothesis that suicidal behaviour and depressive illness may be associated with an altered regulation of adenylyl cyclase.
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PMID:Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims. 813 64

Protein kinase C activity was detected in the cytosolic fraction of quiescent parotid acinar cells; the particulate fraction contained a much smaller proportion of the enzyme. Protein kinase C activity was increased in the membrane fraction and decreased in the cytosol after exposure of intact cells to phorbol 12-myristate 13-acetate (PMA) or the muscarinic-receptor agonist carbachol. The effect of PMA was potentiated by a subthreshold concentration of ionomycin. Immunoblot analysis with anti-protein kinase C antibodies revealed that the protein kinase C-alpha isoform is expressed in rat parotid cells. Other Ca(2+)-dependent isoforms were not detected. Further, agonist stimulation caused the redistribution of protein kinase C-alpha from cytosol to a membrane fraction. Agonists may promote parotid acinar cell activity, including amylase secretion, by increasing the affinity of protein kinase C-alpha for the membrane fraction, presumably via a rise in Ca2+ and diacylglycerol derived from polyphosphoinositide hydrolysis.
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PMID:Translocation of the alpha-isozyme of protein kinase C during stimulation of rat parotid acinar cells by phorbol ester and carbachol. 814 66


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