Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. The inherent instability of TPH has prevented a crystallographic structure from being resolved. For this reason, multiple sequence alignment-based molecular modeling was utilized to generate a full-length model of human TPH. Previously determined crystal coordinates of two highly homologous proteins, phenylalanine hydroxylase and tyrosine hydroxylase, were used as templates. Analysis of the model aided rational mutagenesis studies to further dissect the regulation and catalysis of TPH. Using rational site-directed mutagenesis, it was determined that Tyr235 (Y235), within the active site of TPH, appears to be involved as a tryptophan substrate orienting residue. The mutants Y235A and Y235L displayed reduced specific activity compared to wild-type TPH ( approximately 5 % residual activity). The K(m) of tryptophan for the Y235A (564 microM) and Y235L (96 microM) mutant was significantly increased compared to wild-type TPH (42 microM). In addition, kinetic analyses were performed on wild-type TPH and a deletion construct that lacks the amino terminal autoregulatory sequence (TPH NDelta15). This sequence in phenylalanine hydroxylase (residues 19 to 33) has previously been proposed to act as a steric regulator of substrate accessibility to the active site. Changes in the steady-state kinetics for tetrahydrobiopterin (BH(4)) and tryptophan for TPH NDelta15 were not observed. Finally, it was demonstrated that both Ser58 and Ser260 are substrates for Ca(2+)/calmodulin-dependent protein kinase II. Additional analysis of this model will aid in deciphering the regulation and substrate specificity of TPH, as well as providing a basis to understand as yet to be identified polymorphisms.
J Mol Biol 2000 Sep 29
PMID:Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling. 1099 38

To elucidate the physiological significance of the translocation of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), we investigated substrates of CaM kinase II in the postsynaptic density (PSD). PSD proteins were phosphorylated by CaM kinase II of its PSD complex, and separated by two-dimensional gel electrophoresis. More than 28 proteins were phosphorylated under experimental conditions. Proteins corresponding to CaM kinase II substrates were excised from the gels, eluted electrophoretically, and then sequenced. Several substrates were identified, including PSD95, SAP90, alpha-internexin, neurofilament L chain, cAMP phosphodiesterase, and alpha- and beta-tubulin. Some substrates were also identified by immunoblotting, including N-methyl-D-aspartic acid (NMDA) receptor 2B subunit, 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor 1 (GluR1), neurofilament H chain and dynamin. PSD95, SAP90, dynamin, and alpha-internexin were demonstrated for the first time to be substrates of CaM kinase II. NMDA receptor 2B subunit and GluR1 existed as major substrates in the PSD. Moreover, translocation of CaM kinase II was inhibited by phosphorylation of PSD proteins. These results suggest that CaM kinase II plays important roles in the regulation of synaptic functions through phosphorylation of PSD proteins.
Brain Res Mol Brain Res 2000 Sep 30
PMID:Investigation of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II translocated to the postsynaptic density. 1100 Apr 84

We investigated the mechanisms underlying the increase in diazepam binding inhibitor (DBI) and its mRNA expression induced by nicotine (0.1 microM) exposure for 24 h using mouse cerebral cortical neurons in primary culture. Nicotine-induced (0.1 microM) increases in DBI mRNA expression were abolished by hexamethonium, a nicotinic acetylcholine (nACh) receptor antagonist. Agents that stabilize the neuronal membrane, including tetrodotoxin (TTX), procainamide (a Na(+) channel inhibitor), and local anesthetics (dibucaine and lidocaine), dose-dependently inhibited the increased expression of DBI mRNA by nicotine. The nicotine-induced increase in DBI mRNA expression was inhibited by L-type voltage-dependent Ca(2+) channel (VDCC) inhibitors such as verapamil, calmodulin antagonist (W-7), and Ca(2+)/calmodulin-dependent protein kinase II (CAM II kinase) inhibitor (KN-62), whereas P/Q- and N-type VDCC inhibitors showed no effects. In addition, nicotine exposure for 24 h induced [3H]nicotine binding to the particulate fractions of the neurons with an increased B(max) value and no changes in K(d). Under these conditions, the 30 mM KCl- and nicotine-induced 45Ca(2+) influx into the nicotine-treated neurons was significantly higher than those into non-treated neurons. These results suggest that the nicotine-stimulated increase in DBI mRNA expression is mediated by CAM II kinase activation resulting from the increase in intracellular Ca(2+) through L-type VDCCs subsequent to the neuronal membrane depolarization associated with nACh receptor activation.
Brain Res Mol Brain Res 2000 Sep 15
PMID:Mechanism for increase in expression of cerebral diazepam binding inhibitor mRNA by nicotine: involvement of L-type voltage-dependent calcium channels. 1103 46

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5+/-6.3% and 92. 5+/-11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1+/-0.3% and 8.6+/-2. 1%, respectively. However, the effect of KN-93 was attenuated (47. 8+/-3.6%) in isoproterenol prestimulated cells. Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.
J Mol Cell Cardiol 2000 Dec
PMID:Phosphorylation of phospholamban at threonine-17 in the absence and presence of beta-adrenergic stimulation in neonatal rat cardiomyocytes. 1111 93

Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.
Brain Res Mol Brain Res 2000 Dec 28
PMID:Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons. 1114 4

Since the expression of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) is regulated during brain development, the developmental change of the enzyme was investigated during the neural differentiation of murine P19 embryonal carcinoma cells. CaM kinase II activity was induced during the differentiation of P19 cells treated with retinoic acid. Expression of the enzyme was induced 2 days after the treatment and maximized at 5 days. The enzyme activity increased about approximately 8-fold. The enzyme protein was shown to differ between differentiated and undifferentiated cells. The delta isoform of CaM kinase II was found as the major isoform in P19 cells by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). A total of four and three alternatively spliced variants of delta isoform were detected in P19 cells by RT-PCR analysis and by immunoblotting, respectively. Although multiple alternatively spliced forms have been reported, the major splice variants of delta isoform in differentiated cells were delta l and delta 9 isoforms, which were specifically detected in differentiated cells. In undifferentiated cells, the major splice variant corresponded to delta 2 isoform. These results indicated that the expression of delta isoform of CaM kinase II was induced, and the splicing pattern of the isoform changed, during neural differentiation. Cell type distinctive changes of splicing pattern of delta isoform were also observed not only during differentiation of cultured neuronal cells, but also during development of rat forebrain and cerebellum.
Brain Res Mol Brain Res 2000 Dec 28
PMID:Induction and alternative splicing of delta isoform of Ca(2+)/calmodulin-dependent protein kinase II during neural differentiation of P19 embryonal carcinoma cells and during brain development. 1114 21

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.
Am J Physiol Lung Cell Mol Physiol 2001 May
PMID:Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II. 1129 May 23

The subunit stoichiometry and symmetry of the neuronal alpha-calmodulin-dependent protein kinase II (alphaCaMKII) is investigated in this report to understand the structural basis of its regulation and mechanism at the molecular level. Two preparations are studied, alphaCaMKII obtained by overexpression in baculovirus-transfected insect cells and CaMKII isolated from rat forebrain. The structures, are studied by electron microscopy and image analysis. Single-particle analysis of individual molecular images reveals a molecule with a circular outline and pronounced 6-fold rotational symmetry of the central part. The central part has an outer radius of approximately 6 nm and is composed of six lobes grouped around a hollow centre. The outer ring extends to approximately 15 nm and consists of 12 apparent domains. These data are interpreted in terms of a three-dimensional model of the alphaCaMKII complex consisting of 12 subunits, each corresponding to a single alphaCaMKII polypeptide chain. The inner ring corresponding to approximately one-third of the molecular mass of the complex is made up of the C-terminal association domains. The 12 association domains are arranged in two concentric hexagonal rings at different axial levels and in rotational register. The outer ring corresponding to the remaining molecular mass of the complex is made up of the 12 N-terminal catalytic domains located at an axial level halfway between the two levels of the association domains. The 6-fold symmetry of stacked association domains may derive from subunit arrangements corresponding to either the C6 or the D6 point group symmetries. The symmetry and the resulting subunit arrangement define the pattern and extent of regulatory autophosphorylation within the alphaCaMKII complex.
J Mol Biol 2001 Apr 20
PMID:Oligomeric structure of alpha-calmodulin-dependent protein kinase II. 1130 1

Diisopropyl phosphorofluoridate (DFP) is a type I organophosphorus compound and produces delayed neurotoxicity (OPIDN) in adult hens. A single dose of DFP (1.7 mg/kg, s.c.) produces mild ataxia in hens in 7-14 days, which develops into severe ataxia or paralysis as the disease progresses. We have previously shown altered expression of several proteins (e.g. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) alpha-subunit, tau, tubulin, neurofilament protein (NF), vimentin, GFAP) and an immediate early gene (e.g. c-fos) in DFP-treated hens. Here we show an increase in protein kinase A (PKA) protein level and activity in the spinal cord at 1-day and 5-days time periods after DFP administration. We also determined the protein levels of protein kinase C (PKC), CaM kinase II and several phosphatases (i.e. phosphatase 1 (PP1), phosphatase 2A (PP2A), phosphatase 2B (PP2B) in the spinal cord of DFP-treated hens after 1, 5, 10, and 20 days). There was increase in CaM kinase II alpha subunit level after 10 and 20 days of treatment, and decrease in PKC level at 1-day and 20-days time periods in spinal cord mitochondria. In contrast, the cerebrum, which is resistant to DFP-induced axonal degeneration, did not show change in PKA and CaM Kinase II levels at any time period DFP post-administration. No alteration was found in the protein levels of PP1, PP2A, and PP2B at any time period. An early induction in PKA, which is an important protein kinase in signal transduction, followed by that of CaM kinase might be contributing towards the development of OPIDN in DFP-treated hens.
Mol Cell Biochem 2001 Apr
PMID:Enhanced activity and level of protein kinase A in the spinal cord supernatant of diisopropyl phosphorofluoridate (DFP)-treated hens. Distribution of protein kinases and phosphatases in spinal cord subcellular fractions. 1145 76

Calcium/calmodulin-dependent protein kinase II containing a nuclear localizing signal (CaMKII-alphaB) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). AIP (myristoylated autocamtide-2-related inhibitory peptide), a specific inhibitor of CaMKII provides neuroprotection against NMDA-mediated neurotoxicity. In this study, gene-arrays were used to investigate which apoptosis-associated genes are altered after exposure to NMDA. The data indicate an increased expression (2-7-fold) of five such genes encoding proteins that could be involved in NMDA induced cell death. The up-regulated genes are: FasL; GADD45; GADD153; Nur77 and TNF-R1. Treatment with AIP blocked their altered expression. The results suggest that multiples genes are involved in NMDA-induced excitotoxicity and that AIP, a specific inhibitor for CaMKII, regulates the expression of these apoptosis-associated genes in the retina.
Brain Res Mol Brain Res 2001 Jul 13
PMID:Characterization of apoptosis-genes associated with NMDA mediated cell death in the adult rat retina. 1145 90


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