Morabito Lab Copyright (c) 2013 University of Massachusetts Medical School All rights reserved. http://escholarship.umassmed.edu/morabito Recent documents in Morabito Lab en-us Wed, 13 Feb 2013 17:19:47 PST 3600 Cyclin-dependent kinase 5 regulates PSD-95 ubiquitination in neurons http://escholarship.umassmed.edu/morabito/7 http://escholarship.umassmed.edu/morabito/7 Wed, 25 Apr 2012 09:53:54 PDT Cyclin-dependent kinase 5 (Cdk5) and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer's, learning and memory, and synapse maturation and plasticity. However, the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the ubiquitin E3 ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a nonproteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with beta-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiquitination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis.

]]> Michael J. Bianchetta et al. Animals Cyclin-Dependent Kinase 5 Female Guanylate Kinase Hippocampus Male Membrane Proteins Mice Mice, Knockout Neurons Organ Culture Techniques Protein Interaction Mapping Ubiquitination Cdk5 Regulates the Ubiquitination of PSD-95 in Neurons http://escholarship.umassmed.edu/morabito/6 http://escholarship.umassmed.edu/morabito/6 Mon, 21 Mar 2011 12:03:26 PDT Citation: Bianchetta M. and Morabito M. Cdk5 regulates the ubiquitination of PSD-95 in neurons. Program No. 141.26/G5. 2010 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2010. Online.

Poster abstract can be viewed on page 254 of linked poster abstract compilation.

Link to meeting website

]]> Maria A. Morabito et al. Cyclin-Dependent Kinase 5 Membrane Proteins Intracellular Signaling Peptides and Proteins Ubiquitination Neurons Receptors, Glutamate PSD-95 Tyrosine Phosphorylation and Pyk2 Binding Are Regulated by Cdk5 http://escholarship.umassmed.edu/morabito/5 http://escholarship.umassmed.edu/morabito/5 Mon, 21 Mar 2011 12:03:23 PDT Bianchetta M. and Morabito M. PSD-95 tyrosine phosphorylation and Pyk2 binding are regulated by Cdk5. Program No.717.11/C51. 2009 Neuroscience Meeting Planner. Chicago, IL: Society for Neuroscience, 2009. Online.

Poster abstract is on page 256 of linked compilation of poster abstracts.

Link to meeting website

]]> Michael Bianchetta et al. Cyclin-Dependent Kinase 5 Membrane Proteins Intracellular Signaling Peptides and Proteins Receptors, Glutamate Phosphorylation Tyrosine Focal Adhesion Kinase 2 Receptors, N-Methyl-D-Aspartate The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner http://escholarship.umassmed.edu/morabito/4 http://escholarship.umassmed.edu/morabito/4 Mon, 21 Mar 2011 07:57:29 PDT Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

]]> Rani Dhavan et al. Actinin Animals Binding Sites Calcium Calcium-Calmodulin-Dependent Protein Kinase Type 2 Calcium-Calmodulin-Dependent Protein Kinases Cells, Cultured Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinases Excitatory Amino Acid Agonists Glutathione Transferase Macromolecular Substances Mice Nerve Tissue Proteins Neurons Protein Binding Protein Structure, Tertiary Protein Subunits Rats Rats, Sprague-Dawley Receptors, Glutamate Receptors, N-Methyl-D-Aspartate Recombinant Fusion Proteins Signal Transduction Two-Hybrid System Techniques Partial rescue of the p35-/- brain phenotype by low expression of a neuronal-specific enolase p25 transgene http://escholarship.umassmed.edu/morabito/3 http://escholarship.umassmed.edu/morabito/3 Mon, 21 Mar 2011 07:57:27 PDT Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca(2+)-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimer's disease and may contribute to A-beta peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35-/- phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.

]]> Holger Patzke et al. Aging Animals Brain Cerebellum Cerebral Cortex Corpus Callosum Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinases Gene Expression Hippocampus Mice Mice, Knockout Mice, Transgenic Nerve Tissue Proteins Neurodegenerative Diseases Neurons Phenotype Phosphopyruvate Hydratase Phosphorylation Signal Transduction Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons http://escholarship.umassmed.edu/morabito/2 http://escholarship.umassmed.edu/morabito/2 Mon, 21 Mar 2011 07:57:26 PDT PSD-95 (postsynaptic density 95) is a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. The N-terminal domain of PSD-95 contains three consensus phosphorylation sites for cyclin-dependent kinase 5 (cdk5), a proline-directed serine-threonine kinase essential for brain development and implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. We report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 in vitro and in vivo, and that this phosphorylation is not detectable in brain lysates of cdk5-/- mice. N-terminal phosphorylated PSD-95 is found in PSD fractions together with cdk5 and its activator, p35, suggesting a role for phosphorylated PSD-95 at synapses. In heterologous cells, coexpression of active cdk5 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels, two functions attributed to the N-terminal domain of PSD-95. Consistent with these observations, the lack of cdk5 activity in cultured neurons results in larger clusters of PSD-95. In cdk5-/- cortical neurons, more prominent PSD-95 immunostained clusters are observed than in wild-type neurons. In hippocampal neurons, the expression of DNcdk5 (inactive form of cdk5) or of the triple alanine mutant (T19A, S25A, S35A) full-length PSD-95 results in increased PSD-95 cluster size. These results identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95. Moreover, these observations support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, thus providing a possible mechanism for rapid changes in density and/or number of receptor at synapses.

]]> Maria A. Morabito et al. Animals COS Cells Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinases Intracellular Signaling Peptides and Proteins Ion Channels Macromolecular Substances Membrane Proteins Mice Mice, Knockout Mutagenesis, Site-Directed Nerve Tissue Proteins Neuronal Plasticity Neurons Phosphorylation Protein Structure, Tertiary Rats Receptors, N-Methyl-D-Aspartate Structure-Activity Relationship Substrate Specificity Synapses Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses http://escholarship.umassmed.edu/morabito/1 http://escholarship.umassmed.edu/morabito/1 Mon, 21 Mar 2011 07:57:24 PDT Amyloid-beta (Abeta) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Abeta oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Abeta in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Abeta(1-40) reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Abeta1(1-40)-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Abeta treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Abeta triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

]]> F. Roselli et al. Amyloid beta-Peptides Animals Calcium Cell Membrane Cells, Cultured Cyclin-Dependent Kinase 5 Down-Regulation Frontal Lobe Glutamic Acid Humans Intracellular Signaling Peptides and Proteins inhibitors Membrane Proteins N-Methylaspartate Nerve Tissue Proteins Neurons Peptide Fragments Proteasome Endopeptidase Complex Rats Rats, Wistar Receptors, AMPA Receptors, N-Methyl-D-Aspartate Solubility Synapses