Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1gamma1 and PP1alpha but not PP1beta. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1gamma1 and PP1alpha from brain extracts and associated poorly with PP1beta. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1beta and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1gamma1 > PP1alpha > PP1beta. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1gamma null mice. In the absence of PP1gamma1, both neurabins showed enhanced association with PP1alpha but not PP1beta. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1gamma1 and PP1alpha to the mammalian synapse.
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PMID:The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits. 1201 25

Spinophilin is enriched in dendritic spines, small protrusions of the postsynaptic membrane along the length of the dendrite that contain the majority of excitatory synapses. Spinophilin binds to protein phosphatase 1 with high affinity and targets it to dendritic spines, therefore placing it in proximity to regulate glutamate receptor activity. Spinophilin also binds to and bundles f-actin, the main cytoskeletal constituent of dendritic spines, and may therefore serve to regulate the structure of the synapse. In this study, we sought to determine the structural basis for the targeting of spinophilin to dendritic spines. Our results show that the actin-binding domain of spinophilin is necessary and sufficient for targeting of spinophilin to dendrites and dendritic spines.
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PMID:The actin-binding domain of spinophilin is necessary and sufficient for targeting to dendritic spines. 1223 Mar 5

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.
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PMID:Phosphorylation of spinophilin modulates its interaction with actin filaments. 1241 92

Expression of recombinant PP1 isoforms with fully authentic properties has proven to be a challenge for several laboratories. In order to circumvent this technical limitation in the investigation of isoform-specific roles for PP1, methods have been developed to analyze specific properties of native PP1 isoforms. The well-documented method of ethanol precipitation of tissue extracts has been used to dissociate phosphatase catalytic subunits from their endogenous regulatory subunits and other cellular proteins. Although very low levels of PP1 and PP2A regulatory subunits are sometimes detected in PPC preparations, they are not associated with their respective catalytic subunits because they do not copurify with the catalytic subunits on microcystin-Sepharose (Bauman & Colbran, not shown). Thus, the PPC preparation represents a mixture of native monomeric phosphatase catalytic subunits (including PP1 isoforms, PP2AC, PP4C, and PP6C) that can be used to analyze their interactions with other proteins. The methods described in this report rely on the availability of highly specific antibodies to PP1 isoforms. The sheep antibodies have previously proven effective for immunoblotting and immunoprecipitation, whereas rabbit antibodies have also been used for immunocytochemistry. This paper documents the use of these antibodies in Far-Western overlay and glutathione-agarose cosedimentation assays to investigate interactions of specific PP1 isoforms with recombinant fragments of PP1-targeting subunits (spinophilin, neurabin and GM). Moreover, covalent coupling of affinity-purified sheep antibodies to agarose provided a means for the immuno-isolation of PP1 beta and PP1 gamma 1 from the PPC preparation. Active catalytic subunits are recovered from the affinity resin using chaotropic agents, permitting for the first time the assessment of the effects of specific targeting subunits on activities of individual native PP1 isoforms. These methods have been used successfully to demonstrate that some PP1-interacting proteins discriminate among the isoforms. The isoform inhibition assays provide a measure of the binding equilibrium in the milieu of the phosphatase assay. For example, while some PP1-binding proteins inhibit native PP1 beta and native PP1 gamma 1 with equivalent potency (e.g., PKA-phosphorylated inhibitor-1), spinophilin, neurabin and GM differentiate between these two isoforms; spinophilin and neurabin fragments inhibit native PP1 gamma 1 approximately 20-fold more potently than they inhibit native PP1 beta (Fig. 4), whereas GM inhibits native PP1 beta more potently than native PP1 gamma 1 (not shown). Moreover, the activity of native PP1 gamma 1 is approximately 100-fold more sensitive to neurabin and spinophilin than is the activity of bacterially-expressed recombinant PP1 gamma 1 (Fig. 4). The interpretation of these inhibition assays is consistent with data obtained in Far-Western overlay (Fig. 2) and glutathione-agarose cosedimentation assays (Fig. 3), which assess more stable interactions of PP1 isoforms. Thus, spinophilin and neurabin selectively bind PP1 gamma 1 over PP1 beta, whereas GM is highly selective for PP1 beta. These data are consistent with previous experiments that showed spinophilin and neurabin are present in PP1 gamma 1 complexes in brain extracts, but not in PP1 beta complexes. Moreover, only PP1 beta has been identified in complexes with GM in muscle extracts, although these data did not exclude the possibility that other isoforms were also present. Presumably, these isoform-selective interactions confer different functions on PP1. In summary, we have developed methods that should prove useful in defining the isoform-selectivity of other PP1-targeting subunits. Moreover, these methods may be employed to identify domains in PP1-interacting proteins that confer isoform specificity. Similar strategies may also be used to explore interactions of protein phosphatase catalytic subunits with other proteins.
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PMID:Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits. 1467 48

Signal transduction in the nervous system depends on kinases and phosphatases, whose localization is regulated by a large group of scaffolding proteins. In particular, protein phosphatase-1 mediates dopamine's actions on a variety of substrates, including glutamate receptors, and this, in turn, depends on the binding of protein phosphatase-1 to its binding protein spinophilin. To better understand spinophilin's role in targeting protein phosphatase-1 within neurons, we used a combination of preembedding immunoperoxidase and postembedding immunogold labeling and electron microscopy to determine the localization of this scaffolding protein in macaque prefrontal cortex. Consistent with previous reports, spinophilin was found predominantly in dendritic spines, but a significant number of labeled dendritic shafts and, less frequently, glia and preterminal axons were also identified. By using the postembedding immunogold method, we further examined the distribution of spinophilin within dendritic spines. Spinophilin immunoreactivity was present throughout the spine, but the density of label was heterogeneous and defined two domains. The highest density of label was associated with the postsynaptic density and the 100 nm immediately subjacent to it. The deeper region of the spine, further than 100 nm from the postsynaptic density, had a lower density of spinophilin label. The distribution of spinophilin reported in this study supports its role in modulating glutamatergic neurotransmission but also suggests the possibility that spinophilin may target protein phosphatase-1 to other sites within the spine or to other neuronal or glial compartments.
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PMID:Subcellular distribution of spinophilin immunolabeling in primate prefrontal cortex: localization to and within dendritic spines. 1469 33

Prefrontal cortical functioning depends on dopaminergic neurotransmission, which in turn depends on a complex signal transduction pathway including protein phosphatase-1 (PP1). Targeted localization of PP1 by the scaffolding proteins, spinophilin and neurabin, is critical for dopaminergic modulation of glutamate neurotransmission. In this study, we report the preparation of an antiserum to neurabin, use it to study the subcellular localization of neurabin and compare that to our previous study of spinophilin, a closely related PP1 scaffold. Neurabin is found predominately in dendritic spines, but is also found in other compartments, including dendrites, axons, terminals and glia. This distribution contrasts with that of spinophilin in that neurabin is found in axon terminals where spinophilin is absent, and in parvalbumin-containing interneuron dendrites there is no significant neurabin though these dendrites contain substantial spinophilin. Within the dendritic spine compartment, however, the two proteins are similarly distributed. Both neurabin and spinophilin are concentrated in spines, and double-labeling reveals that they co-localize in most spines. Furthermore, post-embedding immunogold labeling demonstrates that within a spine, neurabin is distributed in the same pattern as spinophilin, concentrated in the postsynaptic density and the 100 nm just below. These results indicate that neurabin and spinophilin share important similarities and differences in their patterns of distribution. Varying patterns of scaffold localization may play an important role in determining the content and action of signal transduction pathways in different neuronal populations or compartments.
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PMID:Subcellular distribution of neurabin immunolabeling in primate prefrontal cortex: comparison with spinophilin. 1521 98

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.
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PMID:Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin. 1522 88

Estrogen (E) treatment of ovariectomized animals increases dendritic spines and/or synaptic protein expression in the hippocampus of female rats [J Neurosci 12 (1992) 2549; Endocrinology 142 (2001) 1284; Endocrinol Rev 20 (1999) 279; Annu Rev Pharmacol Toxicol 41 (2001) 569], mice [Proc Natl Acad Sci USA 101 (2004) 2185], rhesus monkeys [Proc Natl Acad Sci USA 98 (2001) 8071; Endocrinology 144 (2003) 4734; J Comp Neurol 465 (2003) 540] and hippocampal cells in vitro [J Neurosci 16 (1996) 4059; Neuroscience 124 (2004) 549]. The role of E in hippocampal synaptic structural plasticity in males is less well understood. In the present study, we have used a recently developed technique to count spinophilin immunogold-reactive (Ir) puncta as well as in situ hybridization to compare E effects on spinophilin-Ir and mRNA in gonadectomized female and male rats 48 h after E treatment. Spinophilin is an established spine marker, which interacts with several proteins (including actin and protein phosphatase 1) that are highly enriched in spines [Proc Natl Acad Sci USA 94 (1997) 9956; Proc Natl Acad Sci USA 97 (2000) 9287]. We report that E exerts sex-specific effects on dendritic spinophilin-labeled spines in the CA1 region: E treatment significantly increased spinophilin-Ir puncta, indicative of spines, in females, but led to a decrease in males. Furthermore, while hippocampal spinophilin mRNA changes could have occurred earlier, spinophilin mRNA levels were unchanged after 48 h of E in both males and females. This suggests the possibility that E regulates spinophilin protein expression and or stability within dendrites via post-transcriptional mechanisms.
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PMID:Estradiol affects spinophilin protein differently in gonadectomized males and females. 1531 10

Spinophilin is an actin binding protein that positions protein phosphatase 1 next to its substrates in dendritic spines. It contains a single PDZ domain and has the biochemical characteristics of a cytoskeletal scaffolding protein. Previous studies suggest that spinophilin is present in most spines, but the concentration of spinophilin varies from brain region to region in a manner that does not simply reflect differences in spine density. Here, we show that spinophilin is enriched in the great majority of dendritic spines in cerebral cortex, caudatoputamen, hippocampal formation, and cerebellum, irrespective of regional differences in spinophilin concentration. In addition, spinophilin is present postsynaptic to asymmetrical contacts on interneuronal dendritic shafts. We further show that, in hippocampus and ventral pallidum, spinophilin is occasionally present in dendritic shafts adjacent to gamma-aminobutyric acid-containing contacts. Thus, the functional role of spinophilin may not be exclusively restricted to excitatory synapses and may be significant at a small fraction of inhibitory contacts. These data also suggest that the concentration of spinophilin per spine is variable and is likely regulated by local physiological factors and/or regional influences.
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PMID:Cellular and subcellular distribution of spinophilin, a PP1 regulatory protein that bundles F-actin in dendritic spines. 1551 83

Spinophilin is a protein that binds to protein phosphatase-1 and actin and modulates excitatory synaptic transmission and dendritic spine morphology. We have identified three sites phosphorylated by ERK2 (Ser-15 and Ser-205) and cyclin-dependent PK 5 (Cdk5) (Ser-17), within the actin-binding domain of spinophilin. Cdk5 and ERK2 both phosphorylated spinophilin in intact cells. However, in vitro, phosphorylation by ERK2, but not by Cdk5, was able to modulate the ability of spinophilin to bind to and bundle actin filaments. In neurons and HEK293 cells expressing GFP-tagged variants of spinophilin, imaging studies demonstrated that introduction of a phospho-site mimic (Ser-15 to glutamate) was associated with increased filopodial density. These results support a role for spinophilin phosphorylation by ERK2 in the regulation of spine morphogenesis.
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PMID:Phosphorylation of spinophilin by ERK and cyclin-dependent PK 5 (Cdk5). 1572 59


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