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H Tokumitsu

Publications and source records attributed to H Tokumitsu.

At least 19 recordsLinked to original sources
PubMed1998

Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway.

The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B (PKB; also known as c-Akt and Rac) and phosphorylation and sequestration to protein 14-3-3 of the proapoptotic Bcl-2-family member BAD. A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases. Here we report that Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3. Serum withdrawal induced a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis was largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival was blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signalling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis.

Animals↗
PubMed1998

Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51.

A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

Amino Acid Sequence↗
PubMed1998

[Reassessment of a combination of cerebrospinal fluid scintigraphy and nasal pledget counts in patients with suspected rhinorrhea].

A combination study of cerebrospinal fluid scintigraphy and nasal pledget counts was performed using 37 MBq of 111In-DTPA in 12 patients with suspected rhinorrhea. A pledget was inserted and dwelled in each nasal cavity for 6 hours, with the patient prone during at least 30 minutes. A total of 18 studies was implemented and nasal pledget counting method successfully diagnosed all of CSF rhinorrhea. Diagnosis was possible when pledget counts were greater than 1 kcpm. In patients with persistent, intermittent and occult/no nasal discharge, rhinorrhea was found in 100% (5/5), 60% (3/5), 25% (2/8), respectively. Two cases only exhibited positive scintigraphy. MRI or CT cisternography should be first performed in patients with persistent discharge, but in patients with intermittent/occult discharge pledget counting method might take priority of other diagnostic modalities. In conclusion, nasal pledget counting method is a simple and useful tool for detecting rhinorrhea.

Adolescent↗
PubMed1997

The Ca2+/calmodulin-dependent kinase type IV is involved in the CD5-mediated signaling pathway in human T lymphocytes.

The CD5 receptor on T lymphocytes is involved in T cell activation and T-B cell interactions. In the present study, we have characterized the signaling pathways induced by anti-CD5 stimulation in human T lymphocytes. In T lymphocytes, anti-CD5 co-stimulation enhances the phytohemagglutinin/anti-CD28-induced interleukin-2 (IL-2) mRNA accumulation 1.6-fold and IL-2 protein secretion 2. 2-fold, whereby the up-regulation is mediated at both the transcriptional and post-transcriptional level. The CD5 signaling pathway up-regulates the IL-2 gene expression by increasing the DNA binding and transactivation activity of activator protein 1 but affects none of the other transcription factors like nuclear factor of activated T cells, nuclear factor kappaB, Oct, and CD28-responsive complex/nuclear factor of mitogen-activated T cells involved in the regulation of the IL-2 promoter activity. The CD5-induced increase of the activator protein 1 activity is mediated through the activation of calcium/calmodulin-dependent (CaM) kinase type IV, and is independent of the activation of mitogen-activated protein kinases Jun N-terminal kinase, extracellular signal-regulated kinase, and p38/Mpk2, and calcium/calmodul-independent kinase type II. The expression of a dominant negative mutant of CaM kinase IV in T lymphocytes transfected with an IL-2 promoter-driven reporter construct completely abrogates the response to CD5 stimulation, indicating that CaM kinase IV is essential to the CD5 signaling pathway. In addition, it is demonstrated that calcium/calmodulin-dependent kinase type IV is also involved in the stabilization of the IL-2 transcripts, which is observed after co-stimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with anti-CD5.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Calcium/calmodulin-dependent protein kinase kinase: identification of regulatory domains.

We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions 455RKR, 448ILV, or 443SWT blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants 438VKL and 435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a Kd in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458 at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.

Amino Acid Sequence↗
PubMed1997

Inhibitory cross-talk by cAMP kinase on the calmodulin-dependent protein kinase cascade.

The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which was due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site was identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.

Animals↗
PubMed1997

Risk factors for hemorrhage from gastric fundal varices.

The incidence and the risk factors of hemorrhage from gastric fundal varices (FV) have not been fully evaluated. We therefore conducted a retrospective and prospective study to define the incidence and risk factors for such episodes. We investigated 132 patients with cirrhosis and gastric FV. Of these 132 patients, 15 patients had hemorrhagic FV at the time of enrollment. The clinical characteristics were compared between these patients and those without a first hemorrhage from FV. In the patients who had never previously bled, the incidence and risk factors were prospectively investigated. The size of FV was greater and red-spot on the FV were more prevalent in patients with hemorrhagic FV. Child's status was also more severe in these patients. In the 117 patients who had never bled, 34 hemorrhages from FV occurred during the follow-up period. The cumulative risk for such hemorrhage at 1, 3, and 5 years was 16%, 36%, and 44%, respectively. A multiple regression analysis (Cox's model) revealed the size of varices, red-spot on the FV, and Child's status to be statistically significant, as well as independent predictors for hemorrhage from FV. The endoscopic criteria (size of the largest varix and presence of red-spot), as well as the hepatic functional reserve, provide the most essential information for predicting a hemorrhage from FV. An estimation of the probability for hemorrhage from FV based on Cox's model may therefore be beneficial in the clinical management of patients with high-risk FV.

Adult↗
PubMed1997

Degradation of a novel tripeptide, tert-butoxycarbonyl-Tyr-Leu-Val-CH2Cl, with inhibitory effect on human leukocyte elastase in aqueous solution and in biological fluids.

The stability of tert-butoxycarbonyl-Tyr-Leu-Val-CH2Cl (YLV) with inhibitory effect on human leukocyte elastase was investigated in aqueous solution, alpha-chymotrypsin solution and biological media. In all cases studied here, the degradation was observed as a pseudo-first order reaction. The half-life for the degradation of YLV in an aqueous solution of pH 7.4 at 37 degrees C was 35.9 h. YLV was most stable at about pH 3.8-5.8 and the effect of temperature was explained by the Arrhenius equation. The activation energies of the degradation in aqueous solutions at pH 2.0, 4.8, and 7.4 were 24.6, 22.1 and 23.4 kcal/mol, respectively. The degradation products in aqueous solution were analyzed by HPLC-MS and were estimated as Boc-Tyr-Leu-Val-CH2OH at pH 7.4 and H2N-Tyr-Leu-Val-CH2Cl at pH 2.0. In a bovine pancreas alpha-chymotrypsin solution at 37 degrees C, the half-life of YLV was 15 min at 25.6 micrograms/ml of alpha-chymotrypsin solution. In the rat plasma, the half-life of YLV was 42.4 min (YLV 26.7 micrograms/ml plasma), and in rat liver, lung and spleen homogenates, the degradation rate constants of YLV were 37.6, 10.3 and 23.5 times larger than that in plasma solution, respectively (all fluids containing 5 mg protein/ml). YLV was less stable than nafarelin acetate, secretin, adrenocorticotropic hormone (ACTH) and gonadorelin in an aqueous solution of pH 7.4.

Animals↗
PubMed1997

[Successful TSH suppression therapy with triiodothyronine in a patient with pulmonary metastases from differentiated thyroid carcinoma in the absence of 131I uptake].

A 28-year-old woman was referred to us to undergo 131I therapy who had multiple pulmonary metastases from papillary thyroid carcinoma after total thyroidectomy. There was no increased accumulation of a tracer in the pulmonary metastatic foci on whole-body scanning using a 111 MBq diagnostic dose of 131I. However, the pulmonary metastases were gradually decreased in size, and then clearly reduced 8 months after the start of TSH suppression therapy, which was maintained by T3 instead of T4 to bring down the serum TSH level below 0.1 microU/ml. Reduction rates of the foci were 33-76% on chest X-ray. The reduction was confirmed and no new lesions were found on the serial CT scans. Serum thyroglobulin level was lowered 80 to 25 ng/ml by this suppression therapy and progression of disease was not observed under a 54 months' T3 treatment. Thus, TSH suppression therapy might improve survival of patients with differentiated thyroid cancer.

Adult↗
PubMed1996

Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.

Activating Transcription Factor 2↗
PubMed1996

A dual computed tomography linear accelerator unit for stereotactic radiation therapy: a new approach without cranially fixated stereotactic frames.

PURPOSE: To perform stereotactic radiation therapy (SRT) without cranially fixated stereotactic frames, we developed a dual computed tomography (CT) linear accelerator (linac) treatment unit. METHODS AND MATERIALS: This unit is composed of a linac, CT, and motorized table. The linac and CT are set up at opposite ends of the table, which is suitable for both machines. The gantry axis of the linac is coaxial with that of the CT scanner. Thus, the center of the target detected with the CT can be matched easily with the gantry axis of the linac by rotating the table. Positioning is confirmed with the CT for each treatment session. Positioning and treatment errors with this unit were examined by phantom studies. Between August and December 1994, 8 patients with 11 lesions of primary or metastatic brain tumors received SRT with this unit. All lesions were treated with 24 Gy in three fractions to 30 Gy in 10 fractions to the 80% isodose line, with or without conventional external beam radiation therapy. RESULTS: Phantom studies revealed that treatment errors with this unit were within 1 mm after careful positioning. The position was easily maintained using two tiny metallic balls as vertical and horizontal marks. Motion of patients was negligible using a conventional heat-flexible head mold and dental impression. The overall time for a multiple noncoplanar arcs treatment for a single isocenter was less than 1 h on the initial treatment day and usually less than 20 min on subsequent days. Treatment was outpatient-based and well tolerated with no acute toxicities. Satisfactory responses have been documented. CONCLUSION: Using this treatment unit, multiple fractionated SRT is performed easily and precisely without cranially fixated stereotactic frames.

Brain Neoplasms↗
PubMed1996

Requirements for calcium and calmodulin in the calmodulin kinase activation cascade.

We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1 434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC50 values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation.

Alanine↗
PubMed1996

Effects of endoscopic variceal sclerotherapy on azygos vein blood flow and systemic haemodynamics.

The present study was designed to determine the systemic haemodynamic effects of obliterating oesophageal varices by endoscopic sclerotherapy. We evaluated systemic and splanchnic haemodynamics before and after the first course of sclerotherapy in cirrhotic patients. The baseline cardiac index was significantly correlated with baseline azygos vein blood flow (r = 0.64; P < 0.01) and the azygos vein blood flow and cardiac index significantly decreased (-33% and -16%, respectively; P < 0.01) following sclerotherapy. The systemic vascular resistance index was also increased significantly (+ 20%; P < 0.01) in these patients. Moreover, the per cent change in azygos vein blood flow was directly correlated with that of the cardiac index (r = 0.51; P < 0.03). We conclude from these findings that the obliteration of portosystemic collaterals by sclerotherapy significantly reverses hyperdynamic circulation in such patients via a decrease in cardiac preload. The blood flow of the portosystemic shunt per se is a leading contributor to the hyperdynamic circulation observed in patients with well-developed portal systemic collateral vessels.

Adult↗
PubMed1995

Characterization of a Ca2+/calmodulin-dependent protein kinase cascade. Molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase.

Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) can mediate Ca(2+)-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins. We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca(2+)-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647). Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library. Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family. Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2+/ CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.

Amino Acid Sequence↗
PubMed1995

The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization.

Annexin XI is a newly identified annexin which localizes mainly in the nucleus of rat embryonic fibroblasts. There are no typical nuclear localization signals (NLS) in the molecule. To define the region responsible for its nuclear localization, a series of mutants and chimeric cDNA were constructed. These were transiently expressed in COS-7 cells, and the subcellular distributions of the mutants and chimeric proteins were determined by indirect immunofluorescence microscopy. Wild-type annexin XI was located predominantly within the nucleus. Deletion of the N-terminal tail domain (residues 3-196) changed the distribution of the protein from the nucleus to the cytoplasm whereas deletion of the C-terminal core domain (residues 208-504) still kept the protein sorting to the nucleus. Three other mutants lacking 60-80 amino acids in the N-terminal region (residues 3-61, 61-115, and 115-197, respectively) no longer efficiently imported into the nucleus. Furthermore, Escherichia coli beta-galactosidase polypeptide was efficiently localized to the nucleus only when fused with the whole N-terminal region of annexin XI (residues 1-207), not with part of the N-terminal region. In primary cultured rat hepatocytes, annexin XI was distributed in the cytoplasm but not in the nucleus. These results suggest that the whole N-terminal tail domain of annexin XI is necessary and sufficient for its nuclear localization, and may function as NLS in a cell-type specific manner.

Animals↗
PubMed1995

Phosphorylation of CREB by CaM-kinase IV activated by CaM-kinase IV kinase.

Previous reports have shown that CaM-kinase IV can phosphorylate the transcription factor CREB in vitro on Ser133. Furthermore, transfected CaM-kinase IV can activate CREB-dependent transcription, but at a lower efficiency than the cAMP-kinase. In this paper we examine the kinetics and site-specificity of CREB phosphorylation in vitro by CaM-kinase IV after its phosphorylation and activation by a newly discovered brain CaM-kinase IV kinase. Our results show that activated CaM-kinase IV has the same Km (1-5 microM) for CREB phosphorylation, but the Vmax is about 30-fold higher than with non-activated CaM-kinase IV. Activated CaM-kinase IV still shows specificity for phosphorylation of Ser133, the site necessary for transactivation by CREB. It is likely that the lower efficiency of transcriptional activation by transfected CaM-kinase IV in previous studies was due to the fact that the CaM-kinase IV was not activated by CaM-kinase IV kinase.

Amino Acid Sequence↗
PubMed1995

NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus.

The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino-terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17-residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes.

Amino Acid Sequence↗