Browsing by Author "Salek, Asma B."

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    The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5
    (Elsevier, 2018) Morris, Cameron W.; Watkins, Darryl S.; Salek, Asma B.; Edler, Michael C.; Baucum, Anthony J., II; Biology, School of Science
    Spinophilin is the most abundant protein phosphatase 1 targeting protein in the postsynaptic density of dendritic spines. Spinophilin associates with myriad synaptic proteins to regulate normal synaptic communication; however, the full complement of spinophilin interacting proteins and mechanisms regulating spinophilin interactions are unclear. Here we validate an association between spinophilin and the scaffolding protein, disks large-associated protein 3 (SAP90/PSD-95 associated protein 3; SAPAP3). Loss of SAPAP3 leads to obsessive-compulsive disorder (OCD)-like behaviors due to alterations in metabotropic glutamate receptor (mGluR) signaling. Here we report that spinophilin associates with SAPAP3 in the brain and in a heterologous cell system. Moreover, we have found that expression or activation of group I mGluRs along with activation of the mGluR-dependent kinase, protein kinase C β, enhances this interaction. Functionally, global loss of spinophilin attenuates amphetamine-induced hyperlocomotion, a striatal behavior associated with dopamine dysregulation and OCD. Together, these data delineate a novel link between mGluR signaling, spinophilin, and SAPAP3 in striatal pathophysiology.
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    Does spinophilin play a role in alteration of NMDAR phosphorylation?
    (Office of the Vice Chancellor for Research, 2016-04-08) Salek, Asma B.; McBride, Jonathon; Edler Jr., Michael C.; Baucum II, Anthony J.
    Normal brain function requires proper organization of downstream signaling pathways. This organization can be modulated by protein phosphorylation. Protein phosphorylation is a balance of phosphatases, such as protein phosphatase 1 (PP1), and kinases such as protein kinase A (PKA) and cyclin dependent kinase 5 (CDK5). Proper targeting of these proteins is critical for their normal function and is perturbed in various disease states. Spinophilin is critical in targeting PP1 to various substrates making it important in regulating the phosphorylation state and thus the function of various proteins including glutamate receptors, such as AMPARs and NMDARs. NMDARs are abundant postsynaptic proteins that are critical for normal synaptic communication. It has been reported that NMDAR phosphorylation modulates channel function. Here we aim to understand if spinophilin regulates NMDAR phosphorylation and function as well as the mechanisms by which the spinophilin NMDAR interaction are altered. Specifically, we have found that the presence of spinophilin decreases the abundance of PP1 bound to NMDAR. This affect was not observed when a PP1 binding-deficient spinophilin mutant (F451A) was expressed. Furthermore, activation of endogenous PKA and/or overexpression of PKA catalytic subunit robustly increased the association between spinophilin and GluN1 and C-terminal tail of the GluN2B subunit of the NMDAR. Conversely, these associations are decreased when CDK5 is present. Our future studies will evaluate the role of spinophilin in regulating the phosphorylation state of the NMDAR. Taken together, our data demonstrate that spinophilin can associate with multiple subunits of the NMDAR in HEK293 cells and that protein kinases can biphasically modulate these associations.
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    Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction
    (Hindawi, 2017) Hiday, Andrew C.; Edler, Michael C.; Salek, Asma B.; Morris, Cameron W.; Thang, Morrent; Rentz, Tyler J.; Rose, Kristie L.; Jones, Lisa M.; Baucum, Anthony J., II; Biology, School of Science
    Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.
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    Mechanisms Regulating the Association of Protein Phosphatase 1 with Spinophilin and Neurabin
    (American Chemical Society, 2018-11-21) Edler, Michael C.; Salek, Asma B.; Watkins, Darryl S.; Kaur, Harjot; Morris, Cameron W.; Yamamoto, Bryan K.; Baucum, Anthony J., II; Biology, School of Science
    Protein phosphorylation is a key mediator of signal transduction, allowing for dynamic regulation of substrate activity. Whereas protein kinases obtain substrate specificity by targeting specific amino acid sequences, serine/threonine phosphatase catalytic subunits are much more promiscuous in their ability to dephosphorylate substrates. To obtain substrate specificity, serine/threonine phosphatases utilize targeting proteins to regulate phosphatase subcellular localization and catalytic activity. Spinophilin and its homologue neurabin are two of the most abundant dendritic spine-localized protein phosphatase 1 (PP1) targeting proteins. The association between spinophilin and PP1 is increased in the striatum of animal models of Parkinson's disease (PD). However, mechanisms that regulate the association of spinophilin and neurabin with PP1 are unclear. Here, we report that the association between spinophilin and PP1α or PP1γ1 was increased by CDK5 expression and activation in a heterologous cell system. This increased association is at least partially due to phosphorylation of PP1. Conversely, CDK5 expression and activation decreased the association of PP1 with neurabin. As with dopamine depletion, methamphetamine (METH) abuse causes persistent alterations in dopamine signaling which influence striatal medium spiny neuron function and biochemistry. Moreover, both METH toxicity and dopamine depletion are associated with deficits in motor control and motor learning. Pathologically, we observed a decreased association of spinophilin with PP1 in rat striatum evaluated one month following a binge METH paradigm. Behaviorally, we found that loss of spinophilin recapitulates rotarod pathology previously observed in dopamine-depleted and METH-treated animals. Together, these data have implications in multiple disease states associated with altered dopamine signaling such as PD and psychostimulant drug abuse and delineate a novel mechanism by which PP1 interactions with spinophilin and neurabin may be differentially regulated.
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    Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function
    (Cold Spring Harbor Laboratory, 2021-01-01) Salek, Asma B.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of Science
    N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction.
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    Spinophilin regulates phosphorylation and interactions of the GluN2B subunit of the N-methyl-d-aspartate receptor
    (Wiley, 2019-08-02) Salek, Asma B.; Edler, Michael C.; McBride, Jonathon P.; Baucum, Anthony J.; Pharmacology and Toxicology, School of Medicine
    N-methyl-D-Aspartate receptors (NMDARs) are abundant postsynaptic proteins that are critical for normal synaptic communication. NMDAR channel function is regulated by multiple properties, including phosphorylation. Inhibition of protein phosphatase 1 in hippocampal neurons increases NMDAR activity, an effect abrogated by loss of spinophilin, the major protein phosphatase 1 (PP1)-targeting protein in the postsynaptic density (PSD). However, how spinophilin regulates PP1-dependent NMDAR function is unclear. We hypothesize that spinophilin regulates PP1 binding to the NMDAR to alter NMDAR phosphorylation. Our data demonstrate that spinophilin interacts with the GluN2B subunit of the NMDAR. In HEK293 cells, activation and/or overexpression of protein kinase A increased the association between spinophilin and the GluN2B subunit of the NMDAR. Functionally, we found that spinophilin overexpression decreased PP1 binding to the GluN2B subunit of the NMDAR and attenuated the PP1-dependent dephosphorylation of GluN2B at Ser-1284. Moreover, in P28 hippocampal lysates isolated from spinophilin KO compared to WT mice, there was increased binding of GluN2B to PP1, decreased phosphorylation of GluN2B at Ser-1284, and altered GluN2B protein interactions with PSD-enriched proteins. Together, our data demonstrate that spinophilin decreases PP1 binding to GluN2B and concomitantly enhances the phosphorylation of GluN2B at Ser-1284. The putative consequences of these spinophilin-dependent alterations in GluN2B phosphorylation and interactions on synaptic GluN2B localization and function are discussed.
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    Spinophilin-dependent regulation of GluN2B-containing NMDAR-dependent calcium influx, GluN2B surface expression, and cleaved caspase expression
    (Wiley, 2023) Salek, Asma B.; Claeboe, Emily T.; Bansal, Ruchi; Berbari, Nicolas F.; Baucum, Anthony J., II.; Biology, School of Science
    N-methyl-d-aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor conductance and surface localization, two alterations that can modulate overall channel activity. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates the amount of calcium entering the cell and subsequent activation of calcium-dependent processes. The dendritic spine enriched protein, spinophilin, is the major synaptic protein phosphatase 1 (PP1) targeting protein. Depending on the substrate, spinophilin can act as either a PP1 targeting protein, to permit substrate dephosphorylation, or a PP1 inhibitory protein, to enhance substrate phosphorylation. Spinophilin limits NMDAR function in a PP1-dependent manner. Specifically, we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284 on GluN2B. However, how spinophilin modifies NMDAR function is unclear. Herein, we utilize a Neuro2A cell line to detail that Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs. Moreover, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression, an effect that is independent of Ser-1284 phosphorylation. In hippocampal neurons isolated from spinophilin knockout animals, there is an increase in cleaved caspase-3 levels, a marker of calcium-associated apoptosis, compared with wildtype mice. Taken together, our data demonstrate that spinophilin regulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin enhances neuronal cleaved caspase-3 expression.