PubMed Health⌕ Search

Biomedical subjects

T R Soderling

Publications and source records attributed to T R Soderling.

At least 37 records · Page 2Linked to original sources
PubMed1997

Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells.

The surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross-linking with the membrane-impermeant reagent bis (sulfosuccinimidyl) suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by > 90%, whereas immunoreactivity for the lower band was reduced by only 10-20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.

Animals↗
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

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

Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit.

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B.A., Chang, B.H., & Soderling, T.R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B.A., Ng, L.Y., & Soderling, T.R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.

Amino Acid Sequence↗
PubMed1996

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Amino Acid Sequence↗
PubMed1995

Activation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes.

Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV), a member of the CaM-kinase family involved in transcriptional regulation, is stimulated by Ca2+/CaM but also requires phosphorylation by a CaM-kinase kinase for full activation. In this study we investigated the physiological role of a CaM-kinase cascade in Jurkat T human lymphocytes through antigen receptor (CD3) signaling. Total and Ca(2+)-independent CaM-kinase IV activities were increased 8-14-fold by anti-CD3 antibody. This CD3-mediated activation involved phosphorylation since the immunoprecipitated CaM-kinase IV from stimulated Jurkat cells could be subsequently inactivated in vitro by protein phosphatase 2A. CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells or CD3-negative mutant Jurkat cells could be activated in vitro 10-40-fold by CaM-kinase kinase purified from rat brain or thymus, whereas CaM-kinase IV from CD3-stimulated wild-type Jurkat cells was only activated to 2-3-fold by exogenous CaM-kinase kinase. CaM-kinase IV activation was triggered by Ca2+ acting through calmodulin since activation could also be elicited by ionomycin treatment, and CD3-mediated activation was blocked by the calmodulin antagonist calmidazolium. These data are consistent with a CaM-kinase cascade in which CaM-kinase IV is activated by a CaM-kinase kinase cascade triggered by elevated intracellular calcium in Jurkat cells.

Amino Acid Sequence↗
PubMed1995

Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism.

Ca(2+)-sensitive kinases are thought to play a role in long-term potentiation (LTP). To test the involvement of Ca2+/calmodulin-dependent kinase II (CaM-K II), truncated, constitutively active form of this kinase was directly injected into CA1 hippocampal pyramidal cells. Inclusion of CaM-K II in the recording pipette resulted in a gradual increase in the size of excitatory postsynaptic currents (EPSCs). No change in evoked responses occurred when the pipette contained heat-inactivated kinase. The effects of CaM-K II mimicked several features of LTP in that it caused a decreased incidence of synaptic failures, an increase in the size of spontaneous EPSCs, and an increase in the amplitude of responses to iontophoretically applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. To determine whether the CaM-K II-induced enhancement and LTP share a common mechanism, occlusion experiments were carried out. The enhancing action of CaM-K II was greatly diminished by prior induction of LTP. In addition, following the increase in synaptic strength by CaM-K II, tetanic stimulation failed to evoke LTP. These findings indicate that CaM-K II alone is sufficient to augment synaptic strength and that this enhancement shares the same underlying mechanism as the enhancement observed with LTP.

Animals↗
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

Mutational analysis of Ca(2+)-independent autophosphorylation of calcium/calmodulin-dependent protein kinase II.

Previous studies with synthetic peptides indicate that residues 290-309, corresponding to the calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II interact with the catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J., Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R. (1989) J. Biol. Chem. 264, 4800-4804). In the present study, we attempted to locate the pseudosubstrate motif by generation or removal of potential substrate recognition sequences (RXXS/T) at selected positions using site-directed mutagenesis. Based on previous results, Arg297, Thr305/306, and Ser314 were selected as key residues. Single mutations such as N294S, K300S, A302R, A309R, and R311A were expressed, purified, and characterized. Several of the mutants exhibited decreased binding of and activation by CaM, not surprising since the mutations were within the CaM-binding domain. None of the mutants exhibited enhanced Ca(2+)-independent kinase activity toward exogenous substrate, but the K300S and N294S mutants showed a significant enhancement in the rate and stoichiometry of 32P incorporation during Ca(2+)-independent autophosphorylation. Using two-dimensional peptide mapping and phosphoamino acid analyses, enhanced phosphorylation of the introduced Ser residue was demonstrated in the K300S mutant but not in the N294S mutant. This specific Ca(2+)-independent autophosphorylation of Ser300 is consistent with the hypothesis that Arg297 may occupy the P (-3) position in a pseudosubstrate autoinhibitory interaction with the catalytic core in the nonactivated state of the kinase.

Amino Acids↗
PubMed1995

Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors.

Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.

Amino Acid Sequence↗
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

Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. Role of the autoinhibitory domain.

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927) that regulates the Ca(2+)-dependent activation of its phosphatase activity. Substitution of Arg476 and Arg477 or Asp467 to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were coexpressed with the Ca(2+)-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The Vmax values of both truncation mutants with or without Ca2+ were increased relative to wild-type calcineurin. The increased Ca(2+)-independent activity of CaN420 relative to CaN457 indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN420 had similar high Vmax values with or without Ca2+, but the Km value for peptide substrate was increased 5-fold to 125 microM in the absence of Ca2+. The Km values of all the expressed calcineurin species were increased in the absence of Ca2+. The CaN A or CaN A420 subunits alone have low Vmax and high Km (115 microM) values even in the presence of Ca2+. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca2+ binding to CaM and the B subunit, 2) Ca2+ binding to the B subunit also regulates enzyme activity by lowering the Km of the catalytic subunit for substrate, 3) binding of the B subunit is required for high Vmax values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca2+ acting through both CaM and the B subunit.

Amino Acid Sequence↗
PubMed1995

Identification in the calcineurin A subunit of the domain that binds the regulatory B subunit.

Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca2+ to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit. A purified fusion protein containing residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC50 = 0.1 microM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val349-Phe350 and Phe356-Val357) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or coexpressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn2+/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were coexpressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A subunit bind B subunit and that the mutated hydrophobic residues are essential.

Amino Acid Sequence↗
PubMed1994

Activation mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase.

This manuscript examines the mechanisms by which Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT308 to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca2+/CaM-independent (> 20-fold) CaM-kinase IV activities. This activation reaction required Mg2+/ATP and Ca2+/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in Km for syntide-2 with little effect on Km for ATP or Vmax. CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer. The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His305-Lys321 that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10-fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T.R. (1994) J. Biol. Chem. 269, 15520-15527).

Amino Acid Sequence↗