[Structure and function of PKN].
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Actins↗
Biomedical subjects
H Mukai
Publications and source records attributed to H Mukai.
[The usefulness of MRCP in the initial diagnosis of the biliary and pancreatic diseases compared with ERCP].
The purpose of this study was to determine the effectiveness and the limitation of the MR cholangiopancreatography (MRCP) in the initial diagnosis for the biliary and pancreatic diseases compared with the image of Endoscopic retrograde cholangiopancreatography (ERCP). MRCP showed higher performance toward the discovery of the choledocholithiasis of a diameter 4 mm and more. Furthermore, MRCP reflected a malignant tumor at the constrictive part and the dilated proximal part of the biliary and pancreatic duct in 23 example all. An advantage of ERCP was the ability to carry out biopsies and therapeutic procedures, such as biliary drainage and sphincterotomy, while MRCP was an important diagnostic modality in the work-up of the biliary and pancreatic diseases and could help in planning treatment. MRCP would become the examination method first used, and it would be popularized all the more from now on.
Bile Duct Neoplasms↗
Activin A and all-trans-retinoic acid cooperatively enhanced the functional activity of L-type Ca2+ channels in the neuroblastoma C1300 cell line.
Activin, a member of the transforming growth factor-beta superfamily, regulates various physiological functions. In the present study, we investigated the effect of activin on neuronal differentiation, particularly the functional activity of voltage-dependent Ca2+ channels, in murine neuroblastoma C1300 cells. A slight K(+)-induced increase in the intracellular free Ca2+ ([Ca2+]i) was observed in C1300 cells untreated and treated with either activin A or all-trans-retinoic acid, while treatment with both agents significantly enhanced the increase. The [Ca2+]i increases potentiated by activin A and all-trans-retinoic acid were nearly abolished in the presence of 1.0 mM nickel or in the absence of extracellular Ca2+. Nifedipine (0.1 microM) and omega-conotoxin (1.0 microM), inhibitors of L- and N-type Ca2+ channels, respectively, partially inhibited these responses, however the inhibitory effects of these compounds were not additive. In addition, Bay K 8644, an activator of L-type Ca2+ channels, enhanced the K(+)-induced [Ca2+]i increase. These findings indicated that depolarization evoked the Ca2+ influx, at least in part, through L-type Ca2+ channels in C1300 cells treated with both activin A and all-trans-retinoic acid.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Molecular cloning and characterization of a novel mitochondrial phosphoprotein, MIPP65, from rat liver.
A novel 65-kDa protein (designated MIPP65), which was phosphorylated by PKN in vitro in a manner highly dependent on arachidonic acid, was partially purified from the heat-stable proteins extracted from a 30,000g precipitate of rat liver. The cDNA clones were obtained by polymerase chain reaction using oligonucleotides based on partial amino acid sequences. The complete amino acid sequence deduced from the cDNAs contained two homologous regions with the mitochondrial NADH-ubiquinone oxidoreductase 9-kDa subunit precursor at the amino- and carboxyl-termini, whereas the central region was not related to any known proteins and contained a serine cluster. Northern blotting and immunoblotting analyses indicated that MIPP65 was expressed ubiquitously in rat tissues. Immunofluorescence analysis of the endogenous MIPP65 using polyclonal antiserum against MIPP65 showed a predominantly mitochondrial localization in C6 glioma cells. The recombinant MIPP65 expressed in COS7 cells showed a similar pattern of localization to that in C6 glioma cells. On the other hand, deletion of the amino-terminal region of MIPP65 abrogated such localization, indicating that the amino-terminal region contained a mitochondrial-targeting signal. From [32P]orthophosphate-labeled C6 glioma cells, the endogenous MIPP65 could be immunoprecipitated as a phosphoprotein with antiserum against MIPP65. These results suggest that MIPP65 is a novel mitochondrial phosphoprotein that is a candidate substrate for PKN.
Amino Acid Sequence↗
Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN.
PKN is a serine/threonine protein kinase with a catalytic domain homologous to the protein kinase C family and unique N-terminal leucine zipper-like sequences. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that the regulatory domain of PKN interacted with vimentin. We then examined whether PKN would phosphorylate vimentin in vitro. Vimentin proved to be an excellent substrate for PKN, and the phosphorylation of vimentin by PKN occurred in the head domain with the result of a nearly complete inhibition of its filament formation in vitro. Similar results were also obtained with another type III intermediate filament protein, glial fibrillary acidic protein (GFAP). These results raise the possibility that PKN may regulate filament structures of vimentin and GFAP by domain-specific phosphorylation.
Animals↗
[Malignant bile duct diseases and jaundice].
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Ampulla of Vater↗
Interaction of PKN with alpha-actinin.
PKN is a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein alpha-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and alpha-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with alpha-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type alpha-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type alpha-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type alpha-actinin in a Ca2+-insensitive manner. alpha-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and alpha-actinin lacking the actin-binding domain. In vitro translated full-length alpha-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4, 5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length alpha-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and alpha-actinin.
Actinin↗
Specimen banking at National Institute for Environmental Studies, Japan.
The National Institute for Environmental Studies (NIES) in Japan has had more than fifteen years practical experience in specimen banking. Stored specimens are used for the assessment of long-term trends of pollutants. The use of new analytical techniques facilitates the finding of the pollutants of the past.
Academies and Institutes↗
Oral sebaceous carcinoma: report of a case.
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Adenocarcinoma, Sebaceous↗
Digital fluorescence imaging of elementary steps of neurosteroid synthesis in rat brain glial cells.
With fluorescence microscopic imaging, we have examined Ca2+ signaling, LDL uptake and distribution of cytochrome P450 scc on individual rat brain glial cells in order to investigate the molecular mechanisms of neurosteroid synthesis. Astrocytes and oligodendrocytes were cultured from newborn rat brain. Ca2+ signaling was observed in Calcium Green-1 loaded astrocytes upon neurotransmitter stimulations using video-enhanced microscopy. Upon stimulation of serotonin and glutamate, we observed typically three types of Ca2+ signaling which were Ca2+ oscillations, a transient increase in Ca2+ concentration and Ca2+ oscillations superimposed on a transient Ca2+ increase. On the other hand, histamine and ATP induced only a transient increase in Ca2+ without oscillatory response. Uptake of octadecyl rhodamine (R18) labeled LDL by astrocytes and oligodendrocytes was observed in the time scale of 30 min with confocal laser scanning microscopy. Some localization of LDL in the cytoplasm was observed for astrocytes. For oligodendrocytes, incorporated LDL was distributed over the entire cytoplasmic region of both cell body and multiple branched cell processes. The presence of a significant amount of cytochrome P450 scc was demonstrated with immunofluorescence staining in both astrocytes and oligodendrocytes. The density of P450 scc in both glial cells was suggested to be around 1% of that in bovine adrenocortical fasciculata cells. The results lead to an improved quantitative picture of neurosteroid synthesis in glial cells.
Adrenal Cortex↗
[Clinical significant of malassezia furfur specific IgE antibody in atopic dermatitis].
The clinical significance of AlaSTAT (DPC) Specific IgE for Malassezia furfur (Mf) was evaluated by testing the sera of 147 atopic dermatitis (AD) patients. Patients were from 0 to 52 years old, and the prevalence of positive assay results was analyzed by age, class of reactivity, and degree of face and body symptoms. Evaluations of rhinitis, seborrhea as complications and serum total IgE by RIST were included. The prevalence of Mf specific IgE positive sera was lowest in the 0-2 year age group, and increased swiftly in the 3-9 year age group. The older age is, the higher specific IgE antibody for Mf. The prevalence in the 0-2 year age group was significantly different from the other age groups. Although none of the positive results were above AlaSTAT class 2 in the 0-2 year age group, the positive result above AlaSTAT class 2 increased in the 3-9 year age group and was highest in the older age groups, reaching a peak prevalence at age 20 years. A group of age 20 years or older prevalence was significantly different from the 3-9 and 10-19 years old patients. The prevalence of positive did not correlate with face and body symptoms. Adults were more often Mf positive than children. An increasing prevalence of severe face symptoms in adults did correlate with the increased prevalence of Mf specific IgE in the in sera. The AlaSTAT class 3 and 4 results correlated with more severe face symptoms, and complications by seborrhea. The high AlaSTAT class results also corresponded to high total IgE values.
Adolescent↗
Translocation of PKN from the cytosol to the nucleus induced by stresses.
Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.
3T3 Cells↗
Characterization of the interaction between RhoA and the amino-terminal region of PKN.
The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between PKN and a small GTP-binding protein, RhoA. It was revealed that the region corresponding to the amino acid residues 33-111 in the amino-terminal region of PKN was sufficient to confer the ability to associate with RhoA. Each synthetic peptide fragment corresponding to the amino acid residues 74-93 and 94-113 of PKN inhibited the interaction between PKN and RhoA in the in vitro binding assay, suggesting that this region is important in the association with RhoA. The endogenous and the GAP-stimulated GTPase activity of RhoA was inhibited by the interaction with PKN, suggesting the presence of a regulatory mechanism that sustains the GTP-bound active form of RhoA.
Binding Sites↗
PKN associates and phosphorylates the head-rod domain of neurofilament protein.
PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.
Binding Sites↗
The role of the unique motifs in the amino-terminal region of PKN on its enzymatic activity.
The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between the amino-terminal and carboxyl-terminal region of PKN. It was revealed that the amino-terminal region containing the regulatory domain associated with the carboxyl-terminal catalytic region. A synthetic peptide, corresponding to the amino acid residues of PKN from 39 to 53, with substitution of isoleucine46 with serine was shown to become a potent substrate for PKN, and its wild type synthetic peptide inhibited the phosphorylation by PKN. These results suggest that the amino-terminal region of PKN contains the pseudosubstrate sequence and acts as an autoinhibitory region.
Amino Acid Sequence↗
Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.
The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.
Adaptor Proteins, Signal Transducing↗
Identification of a putative target for Rho as the serine-threonine kinase protein kinase N.
Rho, a Ras-like small guanosine triphosphatase, has been implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) to form stress fibers and focal contacts. The form of RhoA bound to guanosine triphosphate directly bound to and activated a serine-threonine kinase, protein kinase N (PKN). Activated RhoA formed a complex with PKN and activated it in COS-7 cells. PKN was phosphorylated in Swiss 3T3 cells stimulated with LPA, and this phosphorylation was blocked by treatment of cells with botulinum C3 exoenzyme. Activation of Rho may be linked directly to a serine-threonine kinase pathway.
3T3 Cells↗