Browsing by Subject "Cyclin-Dependent Kinase 5"
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Item Cdk5-Dependent Regulation of Neuronal MEK1(2013-01-22) Krishnan, Govind; Benavides, David; Tassin, Tara; Bibb, James A.INTRODUCTION: Cyclin-dependent protein kinase 5 (Cdk5) is a member of the Cdk family that is implicated in many regulatory pathways in post-mitotic neurons. The kinase plays an important role in neuronal development and synaptic transmission; its disregulation contributes to neurodegenerative diseases such as Alzheimer's disease. Cdk5 is involved in the regulation of the Ras-Raf-MEK-ERK signaling pathway. It is hypothesized that Cdk5 serves a neuroprotective role by preventing the prolonged stimulation of ERK from inducing cell cycle reentry and, hence, neuronal apoptosis. In order to address whether Cdk5 phosphorylation of MEK1 affects its in vitro kinase activity, we sought to confirm the Cdk5-phosphorylation site of MEK1. Although it has been previously shown that Cdk5 phosphorylates MEK1-T286, our previous unpublished mass spectometry analysis of in vitro kinase reactions identifies the site as T292. We corroborated this data with Western blot analysis of the in vitro Cdk5 phosphorylation of MEK with phosphate-specific antibodies to sites T286 and T292. METHODS: Glutathione S-transferase (GST)-tagged MEK1 was expressed and purified from E.coli. The tagged protein was run through column fractions, with glutathione beads that would attach to the GST tag, such that GST-MEK1 would be purified out of the bacterial lysates. This was then run through gel electrophoresis to confirm that MEK1 had indeed been purified, by comparing the bands seen in the gels to the known molecular weights of GST-MEK1. The protein concentration was determined using a Bradford assay. Non-radioactive and radioactive Cdk5 kinase assays of MEK1 were then performed. MEK1 was incubated in the absence or presence of Cdk5 with Mg2+/ATP or Mg2+/ATP plus trace amounts of [gamma-32P]-ATP, respectively. The non-radiolabeled kinase reactions were subjected to polyacrylamide gel electrophoresis (PAGE) on a 10-20% acrylamide gradient gel, followed by electrophoretic transfer and Western blot analysis with phospho-T286 and phospho-T292 MEK1 antibodies. Phosphorimages of radiolabeled kinase reactions were generated by PAGE, dried gels and standards were exposed to a phosphorimager screen and analyzed on a phosphorimager. RESULTS: Our results confirm that T292 is indeed the Cdk5 site of MEK1. The identity of this site is important because it is the location at which control can be exercised over the ERK pathway.Item Evaluation and Characterization of Novel Signal Transduction Pathways in Striatum(2008-05-13) Sahin, Bogachan; Bibb, James A.In the mammalian central nervous system, protein kinases and protein phosphatases control the function of myriad target proteins in the pre- and postsynaptic compartments, including other protein kinases and phosphatases, neurotransmitter receptors, ion channels, transporters, metabolic enzymes, transcription factors, cytoskeletal elements, and vesicle-docking proteins. Using biochemical and pharmacological approaches, a number of novel striatal signal transduction pathways were evaluated and characterized in the following studies, with emphasis on protein kinase C-mediated signaling. 1) A known and novel form of mouse Adk encoding splice variants of adenosine kinase, the principal enzyme of adenosine metabolism, were cloned from a mouse brain cDNA library and expressed and purified as recombinant proteins with high enzymatic activity. The tissue distribution of adenosine kinase isoform expression was defined. A polyclonal anti adenosine kinase antibody was generated for further characterization of the enzyme. In vitro protein phosphorylation studies using purified protein kinases and in vivo radioimmunoprecipitation assays using the novel antibody for adenosine kinase indicated, however, that this metabolic enzyme is unlikely to be regulated by phosphorylation. 2) Further studies using a candidate approach demonstrated the regulation of several postsynaptic phosphoproteins by striatal adenosine A2A receptor signaling, including ionotropic glutamate receptor subunits, mitogen-activated protein kinase isoforms, a striatal inhibitor of protein phosphatase 1, a protein phosphatase 1- and actin-binding protein, and the cAMP-response element-binding protein. 3) In parallel studies, inhibitor-1, a protein phosphatase 1 inhibitor activated by cAMPdependent protein kinase, was characterized as a novel protein kinase C substrate in vitro and in vivo. Phosphorylation state-specific antibodies raised against this novel phosphorylation site showed that it is dephosphorylated by protein phosphatase 1 and positively regulated by group I metabotropic glutamate receptors in the striatum. Furthermore, protein kinase C-dependent phosphorylation was shown to reduce the efficiency with which inhibitor-1 serves as a substrate for cAMP-dependent protein kinase in vitro and in vivo. 4) Finally, protein kinase C activation was shown to decrease the level of phosphorylation of cyclin-dependent kinase 5 substrates in the striatum, suggesting a possible role for protein kinase C in regulating cyclin-dependent kinase 5 activity.Item Integrated Regulation of PKA by Fast and Slow Neurotransmission(2016-12-19) Thomas, Rachel Elizabeth; Powell, Craig M.; Bibb, James A.; Huber, Kimberly M.; Sternweis, Paul C.The neuronal cyclin-dependent kinase 5 (Ckd5) has been shown to be a major regulator of key signaling events downstream of dopamine receptors in the brain. One critical example of such a pathway is the cAMP-dependent protein kinase, PKA, which plays a well-established role in synaptic plasticity. In addition, recent studies have implicated PKA in the regulation of mood via the brain's mesocorticolimbic pathway. Using in vitro kinase assays and mass spectrometry (MS), we identified a novel Cdk5 phosphorylation site at amino acid residue thr69 (T69) that is unique to the Type II β isoform of the regulatory subunit of PKA, RIIβ. Upon examination of rat brain lysate, RIIβ is shown to be the predominantly expressed isoform within the central nervous system (CNS); the prefrontal cortex, striatum (both ventral and dorsal), and hippocampus exhibit notably high levels of the protein. Immunohistochemistry (IHC) demonstrates expression of RIIβ in all hippocampus subregions - though interestingly only dendritic projections of the granule cell layer of the dentate gyrus (DG) show staining with a phospho-state specific antibody to the T69 site - and ubiquitously throughout the striatal medium spiny neurons (MSNs). These regions are also rich in Cdk5, and the two proteins display co-localization in the dendrites of cultured striatal MSNs. In acute striatal slices, inhibition of Cdk5 resulted in a decrease in T69 phosphorylation, demonstrating in vivo regulation of the site by Cdk5. Via site-directed mutagenesis we generated a full-length T69D RIIβ phosphomimic protein. Compared with unphosphorylated RIIβ, the T69D phosphomimic was a poorer substrate for the PKA catalytic subunit at an activating "autophosphorylation" site, S114. These results were further supported by pharmacological treatments of acute striatal slices, which demonstrated T69/S114 phosphorylation antagonism. As expected from previous studies suggesting that S114 phosphorylation altered PKAcat-RIIβ interactions, a S114D RIIβ phosphomimic was indeed less efficient at inhibiting PKA catalytic subunit activity. Therefore, we believe that Cdk5-dependent phosphorylation of RIIβ acts to inhibit PKA holoenzyme activity via intramolecular changes. It may also affect spatial regulation of PKA. In an RIIβ overlay assay, the T69D phosphomimic demonstrated reduced binding to an important scaffold protein, A kinase anchoring protein 150 (AKAP150). Therefore, we believe through phosphorylation at the T69 site, Cdk5 influences PKA signaling via inter- as well as intra-molecular mechanisms. In order to functionally assess the role of Cdk5-dependent RIIβ phosphorylation, we developed a small interfering peptide (siP) approach that allows us to selectively target Cdk5-RIIβ interactions. We found that the T69 site was negatively modulated by glutamatergic and dopaminergic transmission in the nucleus accumbens (NAc), and that the RIIβ−siP was able to substitute for the glutamatergic input required for dopamine-dependent sustained striatal PKA activation. Moreover, infusion of the peptide into the NAc of rats boosted in vivo PKA activation. The limbic system is critical for establishing emotional salience of, and guiding behavioral output in response to, stressful stimuli. Following acute or chronic stress, levels of phospho-T69 RIIβ in the NAc were altered. Infusion of the RIIβ-siP into the NAc improved the animals' behavioral stress responses in a PKA-dependent fashion. Together, these data support the role of phospho-T69 RIIβ in modulation of PKA activity, thereby impacting striatal neuronal functioning and stress-related behaviors.Item Protein Phosphatase Inhibitor-1 and Cdk5: Of Molecules and Memory(2007-06-12) Nguyen, Baochan; Bibb, James A.Protein phosphatase inhibitor-1 and cyclin-dependent kinase 5 (Cdk5) have been independently implicated in synaptic plasticity, learning, and memory. We began our studies with the identification, confirmation, and characterization of a novel Cdk5-dependent phosphorylation site (Ser6) on inhibitor-1. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5 and protein phosphatases 2A (PP-2A) and 1 (PP-1), respectively. Additionally, protein phosphatase 2B (PP-2B) contributed to dephosphorylation under conditions of high Ca2+. Functionally, Cdk5-dependent phosphorylation of inhibitor-1 intramolecularly impaired dephosphorylation and deactivation of the protein, placing the activities of Cdk5 and protein kinase A (PKA) in synergism in the negative regulation of PP-1. These studies uncovered a potential new regulatory mechanism for Cdk5. Investigation revealed that depolarization differentially regulates the Cdk5-dependent sites of inhibitor-1 and its homologue dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in a cofactor- and N-methyl-D-aspartate (NMDA) receptor-independent manner. Effects on DARPP-32 were Ca2+-mediated and PP-2A-dependent, while effects on inhibitor-1 were nonselectively cation-mediated and either partially PP-2B-dependent or independent of the major serine/threonine phosphatases, depending on the site. Given the uncertain role of inhibitor-1 in learning and memory, we next focused on identifying behaviors and substrates impacted by inhibitor-1 function. Mice constitutively lacking inhibitor-1 displayed enhanced neurogenesis and mildly impaired habituation, but normal contextual fear and novelty learning. Furthermore, levels of hippocampal inhibitor-1 were increased by voluntary wheel running, a stimulus for neurogenesis. Thus, inhibitor-1 may function in an anti-neurogenic mechanism and be more important in the direct or indirect modulation of dopamine-dependent behaviors than in the mnemonic functions of the hippocampus. Using a whole-cell patch clamp approach, we also attempted to identify electrical properties of dentate granule cells that might be affected by Cdk5-dependent phosphorylation of inhibitor-1. Most promising among the results was a reduction in the ability of granule cells lacking inhibitor-1 to faithfully respond to high-frequency trains of stimuli. Granule cell excitability was also increased by pharmacological inhibition of Cdk5 with roscovitine. Finally, in a related study, we helped firmly establish a role for Cdk5 in hippocampal synaptic plasticity by demonstrating that conditional loss of Cdk5 enhances NMDA receptor-mediated currents, particularly of the NR2B type.Item The Regulation and Function of Drosophila Acinus(2017-12-01) Tyra, Lauren Katherine; Johnson, Jane E.; Smith, Dean P.; Pan, Duojia; Krämer, HelmutAutophagy and growth control are two processes critical to organisms that mutually antagonize and regulate on each other. Several well known connections between these processes have been described, but here I describe a new link. Using Drosophila melanogaster as a model system, my thesis research has identified Acinus and Atg1, already known for their functions in autophagy, as growth suppressors. Further, my data show that this suppression is, at least in part, mediated by Atg1 phosphorylating and thereby inhibiting the pro-growth transcriptional co-activator Yorkie. Genetic gain- and loss-of function experiments indicate that this Atg1 function depends on Acinus. This work provides a new role for Atg1 in inhibition of growth and thereby adding a new regulatory pressure on Yorkie. Furthermore, my data indicate that Acinus’ function in promoting basal autophagy is based on its starvation-independent activation of the Atg1 kinase.Item Regulation of Excitatory Neurotransmission, Synaptic Plasticity, and Learning by Cyclin-Dependent Kinase 5(2009-06-17) Hawasli, Ammar Hamami; Bibb, James A.Cyclin-dependent kinase 5 has been implicated in many physiological and pathological processes in the central nervous system. To better understand Cyclin-dependent kinase 5's roles in the adult brain, we developed and studied several conditional Cyclin-dependent kinase 5 knockout model systems. Soon after conditional loss of Cyclin-dependent kinase 5, mice displayed improved hippocampal learning and enhanced synaptic plasticity in the hippocampal Schaffer collateral pathway. The genetically enhanced mice displayed increased N-methyl-D-aspartate receptor-mediated currents and elevated levels of the NR2B N-methyl-D-aspartate receptor subunit. The enhancement in synaptic plasticity was directly attributed to the increased current through NR2B-containing receptors. NR2B levels were elevated in Cyclin-dependent kinase 5 knockout mice due to an impairment in the calpain-mediated degradation of NR2B. Consistently, Cyclin-dependent kinase 5 directly facilitated the degradation of NR2B cytoplasmic-tail in vitro. Cyclin-dependent kinase 5, NR2B, and calpain coimmunoprecipitated in vivo and directly bound one another in vitro. NR2B inhibited Cyclin-dependent kinase 5 activity in vitro, indicating a potential feedback mechanism. These findings suggested that Cyclin-dependent kinase 5 interacts directly with NR2B and calpain to facilitate the degradation of NR2B, thereby attenuating synaptic plasticity. In addition to regulating functional plasticity, Cyclin-dependent kinase 5 also plays roles in structural plasticity and presynaptic function. Cyclin-dependent kinase 5 facilitated the calpain-mediated degradation of spectrin in vitro. Spectrin degradation and depolymerized actin levels were decreased in conditional Cyclin-dependent kinase 5 knockout hippocampus. These results implicate Cyclin-dependent kinase 5 dendritic in spine dynamics which is critical for synaptic plasticity. Loss of Cyclin-dependent kinase 5 also led to a presynaptic enhancement in post-tetanic potentiation and a deficit in paired-pulse facilitation, which are consistent with an increase in probability of synaptic vesicle release, due to increased numbers of vesicles in the readily releasable pool or altered sensitivity to presynaptic calcium. Finally, chronic Cyclin-dependent kinase 5 loss produced increases in behavioral and neuronal excitability followed by electrographic abnormalities in vivo and reduced brain weight. These findings suggest that the enhancement in excitatory neurotransmission which initially led to improvements in learning and plasticity preceded excessive excitability and subsequent neuropathology. Consequently, Cyclin-dependent kinase 5 regulates excitatory neurotransmission, synaptic plasticity.Item The Role of Cdk5 in the Regulation of Dopamine Neurotransmission, Neuronal Excitability, and Reward-Related Behavior(2010-05-14) Benavides, David Roger; Bibb, James A.Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and synaptic plasticity and has been implicated as a homeostatic target of chronic psychostimulant exposure. In order to investigate the role of Cdk5 in the modulation of signal transduction pathways that regulate motivation and rewardrelated behavior, we developed several Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Loss of Cdk5 in the adult brain increased the psychomotor-activating properties of cocaine and enhanced incentive motivation for food. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in conditional Cdk5 knock-out mice. Targeted deletion of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement. In a candidate search for putative Cdk5 substrates that may regulate dopamine-cAMP dependent kinase (PKA) signaling, we identified, confirmed, and characterized Cdk5-dependent phosphorylation of Thr69 RIIβ. We generated novel phosphorylation state-specific antisera to pThr69 RII-beta and found that pThr69 RII-beta levels were reduced in conditional Cdk5 knock-out mice. Functionally, phosphorylation of Thr69 dramatically enhanced calpain-mediated cleavage of RII-beta in vitro, but did not alter inhibition of PKAcat or cAMP binding. Phosphorylation of Thr69 also increased the interaction of RII-beta with an A-kinase anchoring protein (AKAP) docking motif in vitro. In addition, we generated novel phosphorylation state specific antibodies to regulatory phosphorylation sites on cAMP-specific phosphodiesterase 4 (PDE4), an important modulator of PKA signaling. These antibodies to Ser53 PDE4D3 (PKA site) and pSer573 PDE4D3 (ERK site) provide insight into the crosstalk between PKA and ERK pathways in the regulation of cAMP. Finally, we identified and confirmed Thr292 as the site of Cdk5-dependent phosphorylation of MEK1. In conclusion, Cdk5 likely regulates PKA signaling through numerous pathways, including but not limited to DARPP-32, RII-beta, PDE, and MEK1. In summary, these studies identify novel regulatory mechanisms through which Cdk5 controls PKA signaling as well as the interplay between PKA and ERK signaling pathways that regulate motivation and reward-related behavior.