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T R Soderling

Publications and source records attributed to T R Soderling.

At least 19 recordsLinked to original sources
PubMed2001

An important role of neural activity-dependent CaMKIV signaling in the consolidation of long-term memory.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) has been implicated in the regulation of CRE-dependent transcription. To investigate the role of this kinase in neuronal plasticity and memory, we generated transgenic mice in which the expression of a dominant-negative form of CaMKIV (dnCaMKIV) is restricted to the postnatal forebrain. In these transgenic mice, activity-induced CREB phosphorylation and c-Fos expression were significantly attenuated. Hippocampal late LTP (L-LTP) was also impaired, whereas basic synaptic function and early LTP (E-LTP) were unaffected. These deficits correlated with impairments in long-term memory, specifically in its consolidation/retention phase but not in the acquisition phase. These results indicate that neural activity-dependent CaMKIV signaling in the neuronal nucleus plays an important role in the consolidation/retention of hippocampus-dependent long-term memory.

Amino Acid Substitution↗
PubMed2001

Calcium/calmodulin-dependent protein kinase II inhibitor protein: localization of isoforms in rat brain.

A second isoform of Ca2+/calmodulin-dependent-kinase II inhibitor protein (CaM-KIIN) has been identified using the yeast two-hybrid screen. The 1.8kb message encodes a 78 residue CaM-KIINalpha that is 65% identical in its putative open-reading frame and 95% identical in its inhibitory domain to the previously characterized CaM-KIINbeta. CaM-KIINalpha exhibits inhibitory properties towards recombinant mouse CaM-kinase IIalpha indistinguishable from CaM-KIINbeta. The 27 amino acid inhibitory peptide (CaM-KIINtide) derived from CaM-KIIN has the ability to inhibit brain CaM-kinase II activity from multiple organisms including rat, Drosophila and goldfish. Northern analysis of various rat tissues indicates that CaM-KIINalpha is specific to brain whereas CaM-KIINbeta message is also present in testis. In situ hybridization shows a general distribution of both isoforms in rat brain with stronger localization of CaM-KIINbeta in cerebellum and hindbrain and CaM-KIINalpha in frontal cortex, hippocampus and inferior colliculus. An antibody that recognizes both isoforms shows a distribution of CaM-KIIN in rat brain that correlates with immunoreactivity of CaM-kinase II. In cultured mature hippocampal neurons, CaM-KIIN is present in cell bodies and dendrites but, unlike CaM-kinase II, does not display punctate staining at synapses. These results suggest a localized function for CaM-KIIN in inhibiting specialized pools of CaM-kinase II.

Amino Acid Sequence↗
PubMed2000

CaM kinase IV regulates lineage commitment and survival of erythroid progenitors in a non-cell-autonomous manner.

Developmental functions of calmodulin-dependent protein kinase IV (CaM KIV) have not been previously investigated. Here, we show that CaM KIV transcripts are widely distributed during embryogenesis and that strict regulation of CaM KIV activity is essential for normal primitive erythropoiesis. Xenopus embryos in which CaM KIV activity is either upregulated or inhibited show that hematopoietic precursors are properly specified, but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of CaM KIV activity causes commitment of hematopoietic precursors to myeloid differentiation at the expense of erythroid differentiation, on the other hand, constitutive activation of CaM KIV induces erythroid precursors to undergo apoptotic cell death. These blood defects are observed even when CaM KIV activity is misregulated only in cells that do not contribute to the erythroid lineage. Thus, proper regulation of CaM KIV activity in nonhematopoietic tissues is essential for the generation of extrinsic signals that enable hematopoietic stem cell commitment to erythroid differentiation and that support the survival of erythroid precursors.

Amino Acid Sequence↗
PubMed2000

Postsynaptic protein phosphorylation and LTP.

Prolonged changes in synaptic strength, such as those that occur in LTP and LTD, are thought to contribute to learning and memory processes. These complex phenomena occur in diverse brain structures and use multiple, temporally staged and spatially resolved mechanisms, such as changes in neurotransmitter release, modulation of transmitter receptors, alterations in synaptic structure, and regulation of gene expression and protein synthesis. In the CA1 region of the hippocampus, the combined activation of SRC family tyrosine kinases, protein kinase A, protein kinase C and, in particular, Ca2+/calmodulin-dependent protein kinase II results in phosphorylation of glutamate-receptor-gated ion channels and the enhancement of subsequent postsynaptic current. Crosstalk between these complex biochemical pathways can account for most characteristics of early-phase LTP in this region.

Animals↗
PubMed2000

CaM-kinases: modulators of synaptic plasticity.

Calcium signaling is crucial for several aspects of plasticity at glutamatergic synapses, and studies over the past two to three years have identified key functions for Ca(2+)/calmodulin-dependent protein kinases II and IV (CaM-KII and CaM-KIV). Sustained activation of CaM-KII localized at the postsynaptic density results in phosphorylation of numerous synaptic substrates including ion channels, other signaling molecules and scaffolding proteins, to modulate synaptic transmission within minutes. More prolonged responses may be effected through enhanced dendritic protein synthesis of CaM-KII and regulation of nuclear gene transcription by CaM-KIV.

Animals↗
PubMed1999

Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain.

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Amino Acid Sequence↗
PubMed1999

Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors.

The ability of central glutamatergic synapses to change their strength in response to the intensity of synaptic input, which occurs, for example, in long-term potentiation (LTP), is thought to provide a cellular basis for memory formation and learning. LTP in the CA1 field of the hippocampus requires activation of Ca2+/calmodulin-kinase II (CaM-KII), which phosphorylates Ser-831 in the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor (AMPA-R), and this activation/phosphorylation is thought to be a postsynaptic mechanism in LTP. In this study, we have identified a molecular mechanism by which CaM-KII potentiates AMPA-Rs. Coexpression in HEK-293 cells of activated CaM-KII with GluR1 did not affect the glutamate affinity of the receptor, the kinetics of desensitization and recovery, channel rectification, open probability, or gating. Single-channel recordings identified multiple conductance states for GluR1, and coexpression with CaM-KII or a mutation of Ser-831 to Asp increased the contribution of the higher conductance states. These results indicate that CaM-KII can mediate plasticity at glutamatergic synapses by increasing single-channel conductance of existing functional AMPA-Rs or by recruiting new high-conductance-state AMPA-Rs.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗
PubMed1999

The Ca-calmodulin-dependent protein kinase cascade.

The Ca2+-calmodulin-dependent protein kinase (CaM kinase) cascade includes three kinases: CaM-kinase kinase (CaMKK); and the CaM kinases CaMKI and CaMKIV, which are phosphorylated and activated by CaMKK. Members of this cascade respond to elevation of intracellular Ca2+ levels and are particularly abundant in brain and in T cells. CaMKK and CaMKIV localize both to the nucleus and to the cytoplasm, whereas CaMKI is only cytosolic. Nuclear CaMKIV regulates transcription through phosphorylation of several transcription factors, including CREB. In the cytoplasm, there is extensive cross-talk between CaMKK, CaMKIV and other signaling cascades, including those that involve the cAMP-dependent kinase (PKA), MAP kinases and protein kinase B (PKB; also known as Akt). Activation of PKB by CaMKK appears to be important in protection of neurons from programmed cell death during development.

Apoptosis↗
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

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission.

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.

Animals↗
PubMed1998

Characterization of a calmodulin kinase II inhibitor protein in brain.

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII alpha and beta and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i. e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.

Amino Acid Sequence↗
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

Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation.

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca2+- and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.

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

Differential surface expression and phosphorylation of the N-methyl-D-aspartate receptor subunits NR1 and NR2 in cultured hippocampal neurons.

The trafficking and phosphorylation of the NR1 and NR2 subunits of the N-methyl-D-aspartate-type glutamate receptor complex were studied in cultured rat hippocampal neurons. Surface expression was examined by modifying surface receptors via treatment of intact neurons with either the protease chymotrypsin or the cross-linking reagent bis(sulfosuccinimidyl)suberate, followed by quantification of anti-NR1 and anti-NR2B Western blot immunostaining. These studies revealed that only 40-50% of total NR1 immunoreactivity is found at the cell surface, as compared to more than 90% of total NR2B immunoreactivity. Metabolic labeling of the cultures with 32P revealed that NR2 subunits are highly phosphorylated under basal conditions, whereas basal phosphorylation of NR1 subunits is barely detectable. Following stimulation of the cultures with glutamate/glycine or phorbol esters, NR1 phosphorylation was found to be enhanced by 3-5-fold, whereas phosphorylation of NR2 subunits was enhanced by less than 2-fold. To determine whether the difference in the basal NR1 versus NR2 phosphorylation could be due to tyrosine phosphorylation of NR2, phosphoamino acid analyses of NR2 were performed. These analyses revealed phosphorylation on serine but not on threonine or tyrosine; immunoprecipitation and deglycosylation experiments using anti-phosphotyrosine antibodies confirmed that NR2 subunits in the primary hippocampal cultures are not detectably phosphorylated on tyrosine residues. These results demonstrate that the NR1 and NR2 subunits, which assemble into heteromeric complexes to form functional N-methyl-D-aspartate receptors, are trafficked in neurons with differential efficiency to the plasma membrane and exhibit different levels of basal versus stimulated serine phosphorylation.

Animals↗
PubMed1997

Quantitation of AMPA receptor surface expression in cultured hippocampal neurons.

Protein and messenger RNA levels of the AMPA-type glutamate receptor subunits 1-3 are high in many brain regions, but it is not known how much of the glutamate receptor protein is expressed on the surface of neurons in the form of functional receptors. To provide insight into this matter, western blot immunoreactivities for glutamate receptors 1 and 2/3, as well as binding of the specific ligand [3H]AMPA, were quantified following three independent treatments modifying surface receptors in intact primary hippocampal cultures: (i) proteolysis of surface receptors by chymotrypsin, (ii) cross-linking of surface receptors with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate, and (iii) biotinylation of surface receptors with the membrane-impermeant reagent sulfosuccinimidyl-2(biotinamido)ethyl-1,3-dithiopropionate. All three of these methods demonstrated that 60-70% of total glutamate receptor subunit 1 protein and 40-50% of total glutamate receptor 2/3 protein are expressed on the surface of hippocampal neurons. Parallel studies revealed that 52% of total [3H]AMPA binding sites could be precipitated with avidin beads following biotinylation of intact cultures, providing an estimate of [3H]AMPA binding site surface expression in accord with the estimates of the surface expression of glutamate receptor subunits 1-3. Experiments examining the surface expression of 32P-labeled glutamate receptor subunit 1 demonstrated that approximately 65% of the phosphorylated form of the subunit is located in the plasma membrane, an estimate similar to the that derived via western blot for the entire glutamate receptor subunit 1 population in the same samples. Moreover, no significant change in the surface expression profile of the glutamate receptor subunits 1-3 was observed following stimulatory treatments known to increase glutamate receptor phosphorylation. These data indicate that slightly more than half of the AMPA receptors in cultured hippocampal neurons are located in the plasma membrane, and that AMPA receptor surface expression is not rapidly altered by glutamate receptor phosphorylation.

Animals↗