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M B Kennedy

Publications and source records attributed to M B Kennedy.

At least 19 recordsLinked to original sources
PubMed1997

Visualization of the distribution of autophosphorylated calcium/calmodulin-dependent protein kinase II after tetanic stimulation in the CA1 area of the hippocampus.

Autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) at threonine-286 produces Ca2+-independent kinase activity and has been proposed to be involved in induction of long-term potentiation by tetanic stimulation in the hippocampus. We have used an immunocytochemical method to visualize and quantify the pattern of autophosphorylation of CaMKII in hippocampal slices after tetanization of the Schaffer collateral pathway. Thirty minutes after tetanic stimulation, autophosphorylated CaM kinase II (P-CaMKII) is significantly increased in area CA1 both in apical dendrites and in pyramidal cell somas. In apical dendrites, this increase is accompanied by an equally significant increase in staining for nonphosphorylated CaM kinase II. Thus, the increase in P-CaMKII appears to be secondary to an increase in the total amount of CaMKII. In neuronal somas, however, the increase in P-CaMKII is not accompanied by an increase in the total amount of CaMKII. We suggest that tetanic stimulation of the Schaffer collateral pathway may induce new synthesis of CaMKII molecules in the apical dendrites, which contain mRNA encoding its alpha-subunit. In neuronal somas, however, tetanic stimulation appears to result in long-lasting increases in P-CaMKII independent of an increase in the total amount of CaMKII. Our findings are consistent with a role for autophosphorylation of CaMKII in the induction and/or maintenance of long-term potentiation, but they indicate that the effects of tetanus on the kinase and its activity are not confined to synapses and may involve induction of new synthesis of kinase in dendrites as well as increases in the level of autophosphorylated kinase.

Animals↗
PubMed1997

The postsynaptic density at glutamatergic synapses.

The postsynaptic density (PSD) is a tiny, amorphous structure located beneath the postsynaptic membrane of synapses in the CNS. Until recently, the molecular composition and function of the PSD were mostly matters of speculation. With the advent of powerful new microchemical tools and molecular-genetic methods, three new classes of proteins have been identified in the PSD at glutamatergic synapses: the PSD-95 family, the NR2B subunit of the NMDA-type glutamate receptor, and densin-180. The PSD-95 family is involved in clustering of NMDA receptors. NR2B is phosphorylated by Ca2(+)-calmodulin-dependent protein kinase type II, a prominent constituent of the PSD. Densin-180 might represent a new class of synaptic adhesion molecule. Study of these molecules is beginning to reveal the functional significance of the PSD.

Animals↗
PubMed1997

Interaction of ion channels and receptors with PDZ domain proteins.

The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo.

Animals↗
PubMed1996

Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

Animals↗
PubMed1996

Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family.

We purified an abundant protein of apparent molecular mass 180 kDa from the postsynaptic density fraction of rat forebrain and obtained amino acid sequences of three tryptic peptides generated from the protein. The sequences were used to design a strategy for cloning the cDNA encoding the protein by polymerase chain reaction. The open reading frame of the cDNA encodes a novel protein of predicted molecular mass 167 kDa. We have named the protein densin-180. Antibodies raised against the predicted amino and carboxyl sequences of densin-180 recognize a 180 kDa band on immunoblots that is enriched in the postsynaptic density fraction. Immunocytochemical localization of densin-180 in dissociated hippocampal neuronal cultures shows that the protein is highly concentrated at synapses along dendrites. The message encoding densin-180 is brain specific and is more abundant in forebrain than in cerebellum. The sequence of densin-180 contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. This arrangement of domains is similar to that of several adhesion molecules, in particular GPIbalpha, which mediates binding of platelets to von Willebrand factor. We propose that densin-180 participates in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses.

Amino Acid Sequence↗
PubMed1996

PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.

PSD-95, a prominent protein component of the postsynaptic density (PSD) fraction from rat forebrain, has been localized by light microscopy to dendrites of hippocampal neurons (Cho et al., 1992) and to the presynaptic plexus of cerebellar basket cells (Kistner et al., 1993). Here we extend these studies to show that an affinity-purified antibody to PSD-95 labels the dendrites of most neurons in the forebrain and of a subset of neurons in the cerebellum. To confirm that PSD-95 is associated with the PSD at forebrain synapses and to clarify whether it is also associated with the presynaptic membrane, we employed immunogold electron microscopy of forebrain synaptosomes. Gold-labeled antibodies to PSD-95 labeled postsynaptic densities in both intact and lysed forebrain synaptosomes but did not label presynaptic terminals or the presynaptic membrane. The asymmetric distribution of PSD-95 at synapses contrasts with that of its homologs, disks-large and ZO-1, which are arranged symmetrically at septate and tight junctions, respectively.

Animals↗
PubMed1996

Visualization of autophosphorylation of Ca2+/calmodulin-dependent protein kinase II in hippocampal slices.

We describe a method for visualizing the relative spatial distribution of autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) in neuronal subcompartments within hippocampal slices. The method employs a monoclonal antibody recognizing only autophosphorylated CaMKII, and an affinity-purified polyclonal rabbit antisera recognizing only nonphosphorylated CaMK II (Patton et al. (1993) Mol. Biol. Cell, 4: 159-172). 50 microns sections cut from fixed 500 microns hippocampal slices are double-labeled with these antibodies bound by secondary antibodies coupled to fluorescein and Cy3, respectively. The distribution of the two antigens in identical optical sections is recorded by dual channel confocal laser scanning microscopy (CLSM). The digital images are analyzed with the program MacPhase to determine the relative levels of staining with antibodies to phosphokinase and antibodies to nonphosphokinase in subcellular domains of neurons. Comparison of data from paired control and experimental slices reveals the spatial distributions of changes in levels of autophosphorylated CaMKII produced by pharmacological treatments. We are able to detect and spatially resolve differences in levels of autophosphorylation of CaMK II between slices subjected to Ca2+ depletion (low autophosphorylation) and slices treated with a phosphatase inhibitor (high autophosphorylation).

Animals↗
PubMed1995

Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.

The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses.

Amino Acid Sequence↗
PubMed1994

The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.

The postsynaptic density (PSD) is a specialization of the submembranous cytoskeleton that is visible in the electron microscope on the cytoplasmic face of the postsynaptic membrane. A subcellular fraction enriched in structures with the morphology of PSDs contains signal-transduction molecules thought to regulate receptor localization and function in the central nervous system. We have purified a prominent tyrosine-phosphorylated glycoprotein of apparent molecular mass 180 kDa, termed PSD-gp180, that is highly enriched in the rat forebrain PSD fraction. The sequences of four tryptic peptides generated from the protein reveal that it is the 2B subunit of the N-methyl-D-aspartate (NMDA) type glutamate receptor. We have confirmed the identity of PSD-gp180 by showing that it reacts with antibodies raised against a unique fragment of the 2B subunit of the NMDA receptor. We also show that the 2B subunit is the most prominently tyrosine-phosphorylated protein in the PSD fraction based upon recognition by an anti-phosphotyrosine antibody. Two types of NMDA receptor subunits have been identified by molecular cloning [Nakanishi, S. (1992) Science 258, 597-603]. The single type 1 subunit is expressed throughout the brain and is necessary for formation of the receptor channel. The four type 2 subunits (2A, 2B, 2C, and 2D) are expressed in discrete brain regions, contain unusually long unique C termini, and confer distinct kinetic properties on NMDA receptors that contain them. Our findings suggest that, in the forebrain, NMDA receptor subunit 2B may serve to anchor NMDA receptors at the postsynaptic membrane through its interaction with the PSD. The prominent presence of tyrosine phosphate further suggests that the NMDA receptor may be regulated by tyrosine phosphorylation or that it may participate in signaling through tyrosine phosphorylation and through its ion channel.

Amino Acid Sequence↗
PubMed1993

The postsynaptic density.

The postsynaptic density is a specialization of the nerve cell's submembrane cytoskeleton that is hypothesized to participate in the regulation of synaptic adhesion, transmitter receptor clustering, and modulation of receptor sensitivity. Until recently, many of the major proteins in the highly insoluble postsynaptic density fraction remained uncharacterized. Modern immunological and microsequencing methods now make it possible to define more precisely the molecular composition and function of this intriguing organelle.

Animals↗
PubMed1993

Autophosphorylation of type II CaM kinase in hippocampal neurons: localization of phospho- and dephosphokinase with complementary phosphorylation site-specific antibodies.

We have visualized the distribution of autophosphorylated type II CaM kinase in neural tissue with the use of two complementary antibodies: a monoclonal antibody that binds to the alpha and beta subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in beta) and affinity-purified rabbit antibodies that bind to both subunits only when they are not phosphorylated at these residues. We used these antibodies to double-label organotypic hippocampal cultures, detecting the mouse monoclonal antibody with rhodamine and the rabbit polyclonal antibodies with fluorescein. In double-exposed photographs, the ratios of intensities of the two fluorophores revealed the relative proportion of autophosphorylated and nonphosphorylated kinase in individual neurons throughout the cultures. We found that autophosphorylated and nonphosphorylated kinase are colocalized throughout most neurons rather than segregated within distinct cells or subcellular domains. However, the variations in intensity of the two fluorophores indicated that the proportion of autophosphorylated kinase is consistently higher in neuronal somas than in the neuropil. Incubation of the cultures in Ca2+ free medium dramatically reduced both the level of autophosphorylated kinase detected biochemically and the relative intensity of fluorescent staining with the phosphokinase specific monoclonal antibody. These results support the hypothesis that regulation of Ca(2+)-independent CaM kinase activity in vivo occurs by a dynamic equilibrium between autophosphorylation and dephosphorylation and that this equilibrium is maintained, at varying steady-state levels, in all parts of neurons.

Amino Acid Sequence↗
PubMed1992

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.

Amino Acid Sequence↗
PubMed1991

Autophosphorylation of type II Ca2+/calmodulin-dependent protein kinase in cultures of postnatal rat hippocampal slices.

Autophosphorylation of Thr286 on type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) in vitro causes kinase activity to become partially independent of Ca2+. Here we report that Thr286 is the major CaM kinase autophosphorylation site occupied in situ in "organotypic" hippocampal cultures. Measurement of Ca(2+)-independent CaM kinase activity revealed that approximately one-third of the kinase is autophosphorylated in situ when the basal Ca2+ concentration is 15-43 nM. This proportion was substantially reduced 30 min after removal of extracellular Ca2+ or treatment of the cultures with protein kinase inhibitors and was increased by treatment with okadaic acid. Therefore, the high proportion of autophosphorylated kinase at basal Ca2+ concentrations appears to be maintained by Ca(2+)-dependent autophosphorylation. Homogenates of intact hippocampi also contain a high proportion of Ca(2+)-independent type II CaM kinase, 13-23% depending on developmental age. Thus, in hippocampal neurons, an important function of the autophosphorylation mechanism may be to produce a relatively high level of CaM kinase activity, even at basal Ca2+ concentrations, permitting both upward and downward local regulation by physiological agents.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

The alpha subunit of type II Ca2+/calmodulin-dependent protein kinase is highly conserved in Drosophila.

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. We have isolated a Drosophila cDNA encoding an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity. The gene encoding the cDNA is located between 102E and F on the fourth chromosome. The protein product of this gene is expressed at much higher levels in the fly head than in the body. Thus, both the amino acid sequence and the tissue specificity of the mammalian kinase are highly conserved in Drosophila.

Amino Acid Sequence↗
PubMed1990

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase.

Brain type II Ca2+/calmodulin-dependent protein kinase was found to phosphorylate smooth muscle myosin, incorporating maximally approximately 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2(+)-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.

Amino Acids↗
PubMed1990

Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain.

It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the alpha subunit and Thr306 and Ser315 in the beta subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the Kact for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.

Adenosine Triphosphate↗