Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.
The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/GQ1D-CY75, author = {Luong, Tinh Nghi}, title = {Signaling Proteins in the Post-Synaptic Density}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/GQ1D-CY75}, url = {https://resolver.caltech.edu/CaltechTHESIS:06012010-124413933}, abstract = {The ability of an organism to respond to its environment stems from synapses and signaling in the post-synaptic density (PSD). Neurological disorders often occur at the level of faulty signal transduction in the PSD. Here we describe the behavioral characterization of Densin 180, a PSD-enriched scaffold protein. We also report on the regulation of Ras guanine release factor1 (RasGRF1), a guanine exchange factor that promotes activation of Ras and thus the ERK pathway as part of an NMDA (N-methyl-D-aspartate) receptor complex with CaMKII (Ca2+/calmodulin-dependent kinase). The Densin KO exhibits severe nest building deficits, elevated anxiety and aggressiveness, impaired sensorimotor gating, hyperlocomotion to novel objects, and short-term memory (hippocampal- and cortical- dependent) deficits. These behavioral abnormalities resemble schizophrenia and autism. Decreases in the schizophrenia susceptibility gene products, DISC1 and mGluR5, are observed in the KO relative to the WT and may be a result of a decrease in their common binding partner α-actinin. α-actinin is known to regulate mGluR5 surface levels. Cross-linking and stabilization of PSD protein architecture by scaffold proteins like Densin may contribute to some of the observed behavioral abnormalities. The Densin KO also has blunted activity-dependent gene expression. Steady-state levels of the immediate early genes Arc and c-fos are decreased in the hippocampus and cortex of brain sections. Levels of Arc induced in response to stimulation by the neurotrophin BDNF is significantly decreased in the Densin KO neurons after 8 hours of treatment. Impairments in BDNF signaling can lead to affective and cognitive disorder and has a role in cortical inhibition. Dysfunction in BDNF signaling and DISC1 signaling have been previously implicated in autism. The Densin KO also is susceptible to seizures, in particular when injected with Nembutal, a GABA(A) agonist. One point of intersection between signaling pathways that involve DISC1, mGluR, and BDNF is at the level of ERK signaling, which if impaired may establish a hypoglutamatergic state in the Densin KO.
In addition to characterizing the Densin KO, we studied possible interactions of RasGRF1 and CaMKII with the NR2B subunit tail of the NMDA receptor. CaMKII phosphorylation sites on RasGRF1 were identified, including Ser916, by mass spectrometry. Immunoprecipitation from HEK cells revealed that RasGRF1 enhances CaMKII association with NR2B.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/5NQ9-KX48, author = {Beale, Holly C.}, title = {Synaptic Signal Transduction and Transcriptional Control}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/5NQ9-KX48}, url = {https://resolver.caltech.edu/CaltechTHESIS:05212010-144632355}, abstract = {Synaptic signal transduction regulates synaptic plasticity, and, on a larger scale, memory itself. The aim of this dissertation is to elucidate some of the mechanisms that control synaptic plasticity in the short term by modulating synaptic morphology and in the long term by controlling gene expression.
One modification associated with synaptic plasticity is the change in the size of the spine, the micron-scale structure on the dendrite which supports the synapse. The size and shape of the spine are controlled by the actin cytoskeleton. I studied how stimulation of synaptic receptors drives changes in activation of proteins that regulate actin polymerization. We identified neuron-specific aspects of a canonical actin regulation pathway and characterized activity-regulated phosphatase activity.
Changes in spine size and other events associated with synaptic plasticity can begin within seconds of synaptic stimuli, but persistent changes require gene expression. For example, Arc, an immediate early gene required for changes in synaptic strength to persist, is the only transcript known to be both transcribed in response to synaptic stimulation and translocated specifically to the site of the stimulation. However, the role of Arc in promoting the plasticity of the synapse is still under investigation. We studied its binding partners and found that an interaction demonstrated in non-neuronal cells was not evident in neurons.
We also studied changes in transcription over longer time periods. In order to identify pathways involving the postsynaptic protein densin, we assessed global changes in transcription with RNA-Seq, which uses ultra-high-throughput, short-read sequencing to measure transcript abundance. Compared to wild-type mice, densin knockout mice exhibit increased abundance of CaMKIIα (a densin binding partner), increased abundance of immediate early gene expression including Arc, and downregulated GABA_AR subunits.
In summary, we investigated posttranslational modifications that take place within seconds of stimulation, binding interactions occurring in steady-state conditions in wild-type mice, and homeostatic adaptations to the chronic absence of a gene. These investigations into synaptic signaling illustrate not only the complexity of synapse-related regulatory networks but also the range of time scales they span.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/HYHX-A061, author = {Medina-Marino, Andrew G. A.}, title = {Construction and Initial Characterization of the Densin Knockout Mouse}, school = {California Institute of Technology}, year = {2009}, doi = {10.7907/HYHX-A061}, url = {https://resolver.caltech.edu/CaltechETD:etd-06022009-194041}, abstract = {Densin-180 is a core protein of postsynaptic densities (PSDs) in excitatory neurons. Densin is known to interact with Maguin-1 and PSD-95, suggesting that it plays a role in the NMDA receptor complex. Densin also interacts with δ-Catenin and N-Cadherin, an adhesion complex known to play a role in spine morphology. A ternary complex of Densin, CaMKII, and alpha-actinin suggests that Densin plays a key role in cytoskeleton dynamics. Finally, Densin can directly bind to shank, a core scaffolding molecule of the postsynaptic density. The association of Densin with such diverse complexes of proteins suggests that it acts as an integrator of numerous signaling cascades. Here I describe the construction and initial characterization of a Densin knockout mouse. Mice homozygous for the Densin deletion are prone to seizures induced by barbiturates. Also, Densin^-/- animals have altered spine morphologies and show changes in the expression levels of other core PSD proteins. Densin^-/- neurons in culture exhibit an overall decrease in their dendritic complexity. Furthermore, we show that in the absence of the NMDA receptor, Densin can act to bind CaMKII in the PSD. A new high-throughput method for studying changes in gene transcription, RNA seq, was also used to study the effect of the Densin deletion on the forebrain and the hippocampus. This work represents the first time RNA seq has been used to study an animal with a knockout mutation. Two candidate genes that may mediate the seizure sensitivity, Npas4 and GABAAα2, were identified by this method. Npas4 is known to directly affect the number of inhibitory synapses formed by neurons, and GABAAα2 is a major GABA receptor subunit that mediates the effects of Nembutal. These results suggest that Densin may play a role in maintaining the balance between inhibitory and excitatory networks. Together, our results demonstrate that Densin is important for dendritic arbor formation, spine morphology, CaMKII localization in the PSD, and seizure susceptibility.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/d0zv-g984, author = {Mihalas, Stefan}, title = {Quantitative Model of Calcium/Calmodulin- Dependent Protein Kinase II Activation}, school = {California Institute of Technology}, year = {2006}, doi = {10.7907/d0zv-g984}, url = {https://resolver.caltech.edu/CaltechETD:etd-06052006-142955}, abstract = {Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key element in the calcium second messenger cascades that lead to long term potentiation (LTP) of synaptic strength. In this thesis, I have constructed kinetic models of activation of CaMKII and measured some of the unknown parameters of the model. I used the models to elucidate mechanisms of activation of CaMKII and to study the kinetics of its activation under conditions similar to those in dendritic spines.
In chapter 2, I developed a new experimental method to rapidly stop the autophosphorylation reaction. I used this method to measure the catalytic turnover number of CaMKII. To quantitatively characterize CaMKII atophosphorylation in nonsaturating calcium, I also measured the autophosphorylation turnover number when CaMKII is activated by calmodulin mutants that can bind calcium ions only in either the amino or the carboxyl lobes.
Previous models of CaMKII activation assumed that binding of calmodulins to individual CaMKII subunits is independent and that autophosphorylation occurs within a ring of 6 subunits. However, a recent structure of CaMKII suggests that pairs of subunits cooperate in binding calmodulin and raises the possibility that the autophosphorylation occurs within pairs of subunits. In chapter 3, I constructed a model in which CaMKII subunits cooperate in binding calmodulin. This model reconciled previous experimental results from the literature that appeared contradictory. In chapter 4, I constructed two models for CaMKII autophosphorylation, in which autophosphorylation can occur either in rings or pairs, and used them to design experiments aimed at differentiating between these possibilities. Previously published measurements and the measurements that I performed are more consistent with autophosphorylation occurring within pairs.
In chapter 5, I constructed a model for simultaneous interactions among calcium, calmodulin, and CaMKII, and I used an automatic parameter search algorithm to fit the parameters for this model. I used it to characterize which of the parameters of calcium transients are critical for CaMKII activation.
This modeling work is part of a continuing effort to realistically model the spatial and temporal aspects of calcium second messenger signaling in dendritic spines.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/v7bp-jb59, author = {Vázquez, Luis Enrique}, title = {SynGAP Controls Synapse Formation by Regulating Spine Development and Morphology}, school = {California Institute of Technology}, year = {2004}, doi = {10.7907/v7bp-jb59}, url = {https://resolver.caltech.edu/CaltechETD:etd-06012004-144341}, abstract = {SynGAP is a brain-specific Ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here we report that spine maturation and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95, and subunits of AMPA-type and NMDA-type glutamate receptors are larger and brighter, and appear in spines of mutant neurons by day 10 in vitro; whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature excitatory postsynaptic currents are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses, with more AMPA receptors in them. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of synGAP with a mutated GAP domain or a deletion of the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine maturation. Thus, the GAP activity of synGAP, as well as its association with PSD-95, is important for normal regulation of spine and synapse maturation in hippocampal neurons.}, address = {1200 East California Boulevard, Pasadena, California 91125}, } @phdthesis{10.7907/ze5z-f533, author = {Apperson, Michelle Louise}, title = {Molecular Analysis of the Postsynaptic Density: Cloning and Characterization of Densin-lSO, a Novel Postsynaptic Density-Associated Adhesion Molecule}, school = {California Institute of Technology}, year = {1996}, doi = {10.7907/ze5z-f533}, url = {https://resolver.caltech.edu/CaltechETD:etd-01052007-083947}, abstract = {The postsynaptic density (PSD) is an electron dense structure just beneath the postsynaptic membrane. Several functions have been proposed for the PSD including regulating receptor number and clustering, anchoring signal transduction molecules at the synapse and mediating adhesion between the presynaptic and postsynaptic membranes. However, little was known about the proteins that make up the PSD until the biochemical purification of a PSD fraction from brain was established in 1974. Since then, several interesting proteins have been localized to the PSD fraction. The most abundant PSD protein is the [alpha] subunit of the type II calcium/calmodulin dependent protein kinase ([alpha]CaMKII). This protein is likely to play a role in the calcium-mediated signal transduction at the synapse that mediates certain forms of synaptic plasticity. Another major PSD protein is PSD-95, a member of the guanylate kinase family (GUK) of proteins. Here, I describe the purification and identification of three additional PSD proteins that comigrate at a molecular weight of 180 kDa on SDS-polyacrylamide gels. First, PSDgp180 is identified as the 2B subunit of the N-methyl-D-aspartate receptor (NR2B). NR2B is a major component of the PSD fraction and binds to PSD-95 in vitro. This interaction may anchor NMDA receptors at the synapse. Next, I report the cloning and characterization of densin-180, a 180 kDa PSD protein with a novel adhesion molecule-like sequence. Densin-180 is a brain-specific sialomucin that is enriched in the PSD fraction and localized to the synapse by immunocytochemistry. In order to study the assembly of PSD proteins at the synapse, I use antibodies against [alpha]CaMKII, PSD-95 and densin-180 for double labeling cultured hippocampal neurons. In these cultures, densin-180 protein is the first marker to be expressed and this early densin-180 expression is in a diffuse membrane pattern along dendrites. When synapse formation begins at about 5 days after plating, the densin-180 protein is clustered at synapses and PSD-95 expression is induced. PSD-95 colocalizes with densin-180 clusters. The [alpha]CaMKII protein is expressed later in synapse formation (7 to 9 days in vitro) and may be a marker of mature excitatory neurons. In the brain, densin-180 is localized to the neuropil regions in a punctate pattern likely to represent synaptic staining. In addition, anti-densin-180 is localized to a specific set of cells and that may represent undifferentiated neurons and small processes that may represent dendritic filopodia. The third 180 kDa PSD protein is citron, a recently identified Rho/Rac binding protein. The citron sequence contains numerous motifs found in signal transduction proteins and a myosin-like coiled coil domain. Citron may be a target for Rho/Racdependent signal transduction at the synapse and may mediate physical stabilization of the postsynaptic density.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/nanb-2p11, author = {Molloy, Sean S.}, title = {A study of the type II CA2+/calmodulin-dependent protein kinase on hippocampal neurons}, school = {California Institute of Technology}, year = {1991}, doi = {10.7907/nanb-2p11}, url = {https://resolver.caltech.edu/CaltechETD:etd-08232007-130039}, abstract = {Characterization of the type II Ca2+/calmodulin-dependent protein (CaM) kinase in vitro has revealed several intriguing physical and biochemical properties, including the induction of Ca2+-independent activity by autophosphorylation. This thesis describes our attempts to determine the importance of autophosphorylation to the regulation of the kinase in hippocampal neurons. In order to study the type II CaM kinase in these neurons, we established long-term cultures of rat hippocampal slices. We used these cultures to address several questions regarding the phosphorylation of the CaM kinase in the intact neurons, namely: 1) is the CaM kinase phosphorylated in the cultures under basal conditions, 2) if so, is phosphate incorporated into the sites previously characterized in vitro, 3) can phosphorylation of the CaM kinase be modulated in the neurons. Incubation of slice cultures with radiolabeled phosphate in situ showed that both the [alpha] and [beta] subunits of the kinase incorporate phosphate under basal conditions in intact neurons. Furthermore, HPLC analysis of tryptic fragments derived from [alpha] subunit radiolabeled in the cultures in situ revealed that the majority of phosphate was incorporated into Thr286 (the site which controls Ca2+-independent activity in vitro). Measurements of Ca2+-independent activity in homogenates showed that approximately one third of the kinase is autophosphorylated and constitutively active in the cultures. The proportion of Ca2+-independent enzyme in the cultures decreased by 80-90% following removal of external Ca2+. Application of the membrane permeant kinase inhibitors H7 and W7 also caused a substantial decrease in Ca2+-independent kinase activity, while the phosphatase inhibitor, okadaic acid, increased the proportion of Ca2+-independent kinase. Therefore, the resting level of Ca2+-independent CaM kinase apparently reflects a balance between continual Ca2+ dependent autophosphorylation and dephosphorylation by phosphatases. Homogenates of rat forebrains and hippocampi also had substantial levels of Ca2+-independent CaM kinase. These results suggest that the autophosphorylation mechanism acts to maintain a relatively high proportion of constitutively active kinase under conditions of low resting Ca2+ in neurons. This finding is in direct contrast to some models of kinase regulation in hippos pal neurons which predicted that the enzyme would only autophosphorylate following prolonged, synaptically driven increases in intracellular Ca2+. Furthermore, these studies indicate that pharmacological agents could either up or down regulate the level of constitutively active CaM kinase locally, at or near the synapse, by affecting the rate of autophosphorylation or dephosphorylation.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/ete7-xp75, author = {Patton, Bruce Lowell}, title = {Autophosphorylation Sites of the Type II Ca²⁺/Calmodulin-Dependent Protein Kinase: Identification, Regulation of Kinase Activity, and Site-Specific Antibodies}, school = {California Institute of Technology}, year = {1991}, doi = {10.7907/ete7-xp75}, url = {https://resolver.caltech.edu/CaltechETD:etd-07232007-144526}, abstract = {Biochemical and immunological approaches have been developed to study the regulation of the rat neuronal type II Ca²⁺/calmodulin-dependent protein kinase (type II CaM kinase) by autophosphorylation. This thesis describes the identification of in vitro autophosphorylation sites on the CaM kinase and their role in regulating the catalytic activity of the CaM kinase. In addition, this thesis describes the development of antibodies against the type II CaM kinase that specifically recognize either the autophosphorylated kinase or the nonphosphorylated kinase.
The autophosphorylation sites on in vitro autophosphorylated type II CaM kinase were identified by tryptic phosphopeptide mapping using reverse phase HPLC to isolate individual autophosphorylation sites. The sequence of the purified phosphopeptides was determined by gas phase microsequencing and compared to the known sequences of the kinase subunits, deduced from the cDNAs encoding them. The rates of site-specific autophosphorylation, or dephosphorylation by protein phosphatases, was compared with the rate of change in the Ca²⁺/calmodulin-dependence of kinase catalytic activity. In the presence of Ca²⁺ and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the α and β subunits of the type II CaM kinase, Thr²⁸⁶ and Thr²⁸⁷, respectively. Phosphorylation of this site correlated with the generation of Ca²⁺-independent catalytic activity. Removal of free Ca²⁺ ion from the autophosphorylation reaction resulted in the autophosphorylation of two pairs of homologous residues, Thr³⁰⁵ and Ser³¹⁴ in the a subunit a and Thr³⁰⁶ and Ser³¹⁵ in the kinase catalytic activity. In the presence of Ca²⁺ and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the β subunit. Ser³¹⁴/³¹⁵ is resistant to dephosphorylation by purified protein phosphatases 1 and 2A. Selective dephosphorylation of the Thr³⁰⁵/³⁰⁶ autophosphorylation site demonstrated that the presence of phosphate on Thr³⁰⁵/³⁰⁶ inhibits Ca²⁺/calmodulin-stimulated catalytic activity. The presence of phosphate on Ser³¹⁴/³¹⁵ slightly decreases the sensitivity of the kinase to Ca²⁺/calmodulin.
Antibodies that bind to the type II CaM kinase at the Thr²⁸⁶/²⁸⁷ autophosphorylation site were produced in note and rabbits by immunization with thiophosphorylated and nonphosphorylated peptide haptens. A monoclonal antibody was obtained that specifically recognized the autophosphorylated type IICaM kinase. The monoclonal antibody recognized the Thr²⁸⁶/²⁸⁷ autophosphorylation site. A polyclonal antisera was obtained that, when affinity purified, specifically recognized the nonphosphorylated type II CaM kinase. Autophosphorylation of type II CaM kinase on Thr²⁸⁷ potently inhibited binding of the polyclonal antibodies. The monoclonal antibody and polyclonal antisera recognized type II CaM kinase in immunocytochemical sections and were used to assess the extent and distribution type II CaM kinase autophosphorylation in organotypic cultures of rat brain hippocampal slices. Double immunofluorescence immunocytochemistry with the antibodies specific for phosphorylated and nonphosphorylated type II CaM kinase indicated that most neurons and dendrites contain a mixture of phosphorylated and nonphosphorylated kinase, in varying proportions. Removal of extracellular Ca²⁺ greatly reduced the immunoreactivity specific for the phosphorylated kinase, implying that the type II CaM kinase phosphorylation state is in dynamic equilibrium in neurons.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/w18z-4r52, author = {Miller, Stephen G.}, title = {Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Characterization of a Brain-Region Specific Isozyme and Regulation by Autophosphorylation}, school = {California Institute of Technology}, year = {1988}, doi = {10.7907/w18z-4r52}, url = {https://resolver.caltech.edu/CaltechTHESIS:03142013-100553837}, abstract = {A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca²⁺ and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.
The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of α and β/β’ subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β’ subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.
The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca²⁺/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca²⁺-independent after autophosphorylation in the presence of Ca²⁺/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca²⁺ These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca²⁺/calmodulin.
The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca²⁺-independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca²⁺/calmodulin.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/30tg-qa85, author = {Erondu, Ngozi Emmanuel}, title = {Regional Distribution and Subcellular Associations of Type II Calcium and Calmodulin-Dependent Protein Kinase in Rat Brain}, school = {California Institute of Technology}, year = {1987}, doi = {10.7907/30tg-qa85}, url = {https://resolver.caltech.edu/CaltechTHESIS:04152019-165931960}, abstract = {Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase.
With the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The unusually high concentration of the kinase in telencephalic regions may confer upon their neurons specialized responses to calcium that are different from those of neurons in lower brain regions.
The association of the kinase with elements of the cytoskeleton was also investigated. The results of this study showed that autophosphorylation causes an increase in the association of the enzyme with taxol-polymerized microtubules and F-actin. This increase in association was reversed by dephosphorylating phosphokinase with protein phosphatase. These results suggest that autophosphorylation could constitute a mechanism for the regulation of the subcellular associations of the Type II CaM kinase by neuronal activity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/a6p9-vd59, author = {Bennett, Mark Knowles}, title = {Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Purification, Characterization and Molecular Cloning}, school = {California Institute of Technology}, year = {1986}, doi = {10.7907/a6p9-vd59}, url = {https://resolver.caltech.edu/CaltechTHESIS:04122019-162032124}, abstract = {A combination of biochemical, immunochemical, and molecular biological techniques have been employed to purify and characterize a rat brain Ca2+/calmodulin-dependent protein kinase. The enzyme, named type II Ca2+/calmodulin-dependent protein kinase (type II CaM kinase), was identified in rat brain homogenates by its ability to phosphorylate site II on the synaptic vesicle associated protein synapsin I.
Type II CaM kinase has been purified 290 fold over crude homogenates and is found to be composed of multiple copies of two different subunits. Both subunits copurify with kinase activity and are coprecipitated with kinase activity by an anti-kinase monoclonal antibody. The two subunits have molecular weights of 50,000 (α) and 58,000/60,000 (β), and are present in a 3:1 α:β ratio. The type II CaM kinase holoenzyme has a sedimentation coefficient of 16.4 S, a Stokes radius of 95 Å, and a calculated molecular weight of 650,000. A dodecameric holoenzyme consisting of 9 α subunits and 3 β subunits has been proposed. The purified type II CaM kinase phosphorylates several substrates, in addition to synapsin I, at a significant rate, and may therefore be responsible for a number of neuronal responses to Ca2+.
The α subunit of type II CaM kinase has a number of biochemical characteristics which are similar to the major protein component of a subcellular fraction which is derived from brain postsynaptic densities (PSDs). A direct comparison between the a subunit of type II CaM kinase and the major PSD protein using immunochemical and biochemical techniques has revealed that they are in fact very similar or identical proteins.
Two approaches have been taken to further characterize the subunits of type II CaM kinase at a molecular level. The first approach has been to isolate cDNA clones which code for the β subunit. A number of clones have been isolated and sequenced. The ammo acid sequence for the β subunit (predicted from the cDNA sequence) is homologous to several other protein kinases. Southern blot analysis with a β subunit cDNA indicates the existence of a type II CaM kinase multigene family. The second approach to the molecular characterization of the type II CaM kinase subunits has been to determine the amino acid sequence of peptides derived from the α subunit. Two regions of α subunit sequence have been determined, and both are found to be homologous to regions of β subunit amino acid sequence deduced from β subunit cDNA clones.
The molecular characterization of neuronal type II CaM kinase in vitro has both provided insight into the possible function of the enzyme in vivo and suggested experimental approaches which may eventually allow its in vivo function to be directly addressed.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, } @phdthesis{10.7907/774r-7520, author = {Lemke, Greg Erwin}, title = {Identification and Characterization of Glial Growth Factor}, school = {California Institute of Technology}, year = {1983}, doi = {10.7907/774r-7520}, url = {https://resolver.caltech.edu/CaltechTHESIS:10182019-171906142}, abstract = {A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts.
Three independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel electrophoresis in SDS.
Three large-scale purifications of GGF, employing a combination of column chromatography steps and preparative electrophoreses, are described. The molecule has been purified to apparent homogeneity from anterior lobes of the bovine pituitary.
Through the use of nucleic acid precursor incorporation assays, GGF has been shown to be markedly mitogenic for rat Schwann cells, astrocytes and fibroblasts, but inactive when assayed on oligodendrocytes or microglia. Electrophoretic analyses suggest that all responsive cell types are stimulated by a single (the same) molecular species. GGF is the only defined mitogen to which rat Schwann cells respond.
Glial growth factor from bovine brain has been found to be indistinguishable from bovine pituitary GGF, as determined by biochemical, immunological and bioactivity criteria. GGF is non-uniformly distributed among bovine brain regions. It is present in brain extracts prepared from a wide variety of vertebrate species.
Purified human platelet-derived growth factor (PDGF) shares many important properties with GGF. PDGF has been shown to be unable to significantly stimulate the division of rat Schwann cells, however, and therefore appears to be distinct.
Observations made in vitro suggest several possible biological roles for GGF in vivo. These are discussed.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Kennedy, Mary B.}, }