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M B Kennedy

Publications and source records attributed to M B Kennedy.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1990

Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D.

Cell cycle-regulated gene expression is essential for normal cell growth and development and loss of stringent growth control is associated with the acquisition of the transformed phenotype. The selective synthesis of histone proteins during the S phase of the cell cycle is required to render cells competent for the ordered packaging of replicating DNA into chromatin. Regulation of H4 histone gene transcription requires the proliferation-specific promoter binding factor HiNF-D. In normal diploid cells, HiNF-D binding activity is regulated during the cell cycle; nuclear protein extracts prepared from normal cells in S phase contain distinct and measurable HiNF-D binding activity, while this activity is barely detectable in G1 phase cells. In contrast, in tumor-derived or transformed cell lines, HiNF-D binding activity is constitutively elevated throughout the cell cycle and declines only with the onset of differentiation. The change from cell cycle-mediated to constitutive interaction of HiNF-D with the promoter of a cell growth-controlled gene is consistent with, and may be functionally related to, the loss of stringent cell growth regulation associated with neoplastic transformation.

Animals↗
PubMed1990

Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Alkaline Phosphatase↗
PubMed1990

Hippocampal neurons predisposed to neurofibrillary tangle formation are enriched in type II calcium/calmodulin-dependent protein kinase.

The microtubule-associated phosphoprotein, tau, is an integral component of paired helical filaments in Alzheimer neurofibrillary tangles (NFT). The mechanism of NFT formation is unknown but aberrant phosphorylation of tau may be contributory. Calcium/calmodulin-dependent protein kinase type II (CaM kinase II), the most abundant kinase in the brain, phosphorylates tau in vitro. We found CaM kinase II immunoreactivity concentrated in human hippocampal pyramidal neurons of CA1 and the subiculum. In Alzheimer's disease (AD) staining intensity of CA1 and subicular neurons is strikingly increased despite NFT formation and neuronal depletion. Enhanced CaM kinase II activity, possibly a result of deafferentation, may contribute to phosphorylation of tau protein leading to NFT deposition and neuronal death in AD.

Aged↗