Mary B. Kennedy

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.

Regulation of brain Type II Ca2+calmodulin-dependent protein kinase by autophosphorylation: A Ca2+-triggered molecular switch

Calcium/calmodulin-stimulated autophosphorylation of a prominent brain calmodulin-dependent protein kinase (Type II CaM kinase) produces dramatic changes in its enzymatic activity. These changes suggest a mechanism by which the kinase could act as a calcium-triggered molecular switch. Incorporation of 3-12 of a possible total of 30 phosphate groups per holoenzyme causes kinase activity toward exogenous substrates as well as autophosphorylation itself to become independent of calcium. Thus, kinase activity could be prolonged beyond the duration of an initial activating calcium signal. The calcium-independent autophosphorylation could further prolong the active state by opposing dephosphorylation by cellular phosphatases.

Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain

The distribution of type II Ca2+/calmodulin-dependent protein kinase has been mapped in rat brain by immunochemical and immunohistochemical methods using an antibody against its alpha-subunit. The concentration of the kinase, measured by radioimmunoassay, varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of the total hippocampal protein, 1.3% of cortical protein, and 0.7% of striatal protein. It is less concentrated in lower brain structures, ranging from about 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The gradient of staining intensity observed in brain sections by immunohistochemistry corroborates this distribution. Neurons and neuropil of the hippocampus are densely stained, whereas little staining is observed in lower brain regions such as the superior colliculus. Within the diencephalon and midbrain, dense staining is observed only in thalamic nuclei and the substantia nigra. The skewed distribution of alpha-subunit appears to be due in part to the occurrence in the cerebellum and pons/medulla of forms of the kinase with a high ratio of beta- to alpha-subunits. However, most of the variation is due to the extremely high concentration of the kinase in particular neurons, especially those of the hippocampus, cortex and striatum. The unusually high expression of the kinase in these neurons is likely to confer upon them specialized responses to calcium ion that are different from those of neurons in lower brain regions.

A Synaptic Ras-GTPase Activating Protein (p135 SynGAP) Inhibited by CaM Kinase II

Ca2+ influx through N-methyl-D-aspartate- (NMDA-) type glutamate receptors plays a critical role in synaptic plasticity in the brain. One of the proteins activated by the increase in Ca2+ is CaM kinase II (CaMKII). Here, we report a novel synaptic Ras-GTPase activating protein (p135 SynGAP) that is a major component of the postsynaptic density, a complex of proteins associated with synaptic NMDA receptors. p135 SynGAP is almost exclusively localized at synapses in hippocampal neurons where it binds to and closely colocalizes with the scaffold protein PSD-95 and colocalizes with NMDA receptors. The Ras-GTPase activating activity of p135 SynGAP is inhibited by phosphorylation by CaMKII located in the PSD protein complex. Inhibition of p135 SynGAP by CaMKII will stop inactivation of GTP-bound Ras and thus could result in activation of the mitogen-activated protein (MAP) kinase pathway in hippocampal neurons upon activation of NMDA receptors.

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.

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.

Spine architecture and synaptic plasticity.

Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using live-imaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.

Regulation of neuronal function by calcium

In the classical picture of brain function, electrical impulses are initiated in sensory organs and spread rapidly down axons, jumping synaptic clefts by neurochemical transmission. Patterns of electrical activity generated in this way integrate information throughout the brain and result in coordinated motor output. Even as this picture of the central role of electrical transmission was emerging in the mid-20th century, the more speculative neuroscientists reasoned that there must be more to it. In order to store information and adapt to a changing environment, neurons must be able to alter their own properties or those of their neighbors, in highly controlled ways, sometimes permanently.

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 ∼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.

Integration of biochemical signalling in spines

Short-term and long-term changes in the strength of synapses in neural networks underlie working memory and long-term memory storage in the brain. These changes are regulated by many biochemical signalling pathways in the postsynaptic spines of excitatory synapses. Recent findings about the roles and regulation of the small GTPases Ras, Rap and Rac in spines provide new insights into the coordination and cooperation of different pathways to effect synaptic plasticity. Here, we present an initial working representation of the interactions of five signalling cascades that are usually studied individually. We discuss their integrated function in the regulation of postsynaptic plasticity.