Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II to regulate its binding to F-actin

Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 08/2004; 90(2):317-24. DOI: 10.1111/j.1471-4159.2004.02491.x
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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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    • "It is a protein phosphatase I (PP-I) interacting and PP-I – activity modulating protein [83,84] that is primarily found in dendritic spines [83,85]. Intraspinal localization of NEB2 and its F-actin binding and bundling capacity were demonstrated to be modulated via its phosphorylation by the Ca2+/calmodulin dependent kinase II (CAMKII) [86] or the protein kinase A (PKA) [87], linking spinophilin action with its responsiveness to NMDA and adrenergic receptor activity [88,89] (Figure 4). The NEB2-mediated effect on F-actin organization within dendritic spines, thus, depends on Ca2+ as well as on cAMP signaling. "
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    ABSTRACT: Disturbed proteostasis as a particular phenotype of the aging organism has been advanced in C. elegans experiments and is also conceived to underlie neurodegenerative diseases in humans. Here, we investigated whether particular changes in non-disease related proteostasis can be identified in the aged mammalian brain, and whether a particular signature of aberrant proteostasis is related to behavioral performance of learning and memory. Young (adult, n = 30) and aged (2 years, n = 50) Wistar rats were tested in the Morris Water Maze (MWM) to distinguish superior and inferior performers. For both young and old rats, the best and worst performers in the MWM were selected and the insoluble proteome, termed aggregome, was purified from the hippocampus as evidence for aberrant proteostasis. Quantitative proteomics (iTRAQ) was performed. The aged inferior performers were considered as a model for spontaneous, age-associated cognitive impairment. Whereas variability of the insoluble proteome increased with age, absolute changes in the levels of insoluble proteins were small compared to the findings in the whole C. elegans insoluble proteome. However, we identified proteins with aberrant proteostasis in aging. For the cognitively impaired rats, we identified a changed molecular circuitry of proteins selectively involved in F-actin remodeling, synapse building and long-term depression: actin related protein 3 (ARP3), neurabin II (NEB2) and IQ motif and SEC7 domain-containing protein 1 (BRAG2). We demonstrate that aberrant proteostasis is a specific phenotype of brain aging in mammals. We identify a distinct molecular circuitry where changes in proteostasis are characteristic for poor learning and memory performance in the wild type, aged rat. Our findings 1. establish the search for aberrant proteostasis as a successful strategy to identify neuronal dysfunction in deficient cognitive behavior, 2. reveal a previously unknown functional network of proteins (ARP3, NEB2, BRAG2) involved in age-associated cognitive dysfunction.
    PLoS ONE 10/2013; 8(9):e75112. DOI:10.1371/journal.pone.0075112 · 3.23 Impact Factor
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    • "Alternatively, CaMKII binding to spinophilin may enhance phosphorylation of the N-terminal domain of spinophilin itself to decrease interactions with F-actin [63], [64]. Also, the binding to spinophilin may target CaMKII to phosphorylate additional SpAPs that can modulate the F-actin cytoskeleton such as Tiam1 or Kalirin-7 [55], [65], [66]. "
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    ABSTRACT: Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1)-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII) directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin in vitro and in vivo. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging.
    PLoS ONE 02/2012; 7(2):e31554. DOI:10.1371/journal.pone.0031554 · 3.23 Impact Factor
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    • "In neurons, spinophilin regulates the actin cytoskeleton and microtubules by direct and indirect interactions ( Grossman et al., 2004 ; Ryan et al., 2005 ; Bielas et al., 2007 ). Although in DCs, actin and its regulation by Rac regulate DC – T cell interactions ( Benvenuti et al., 2004 ), Rac does not seem to be a target for spinophilin or the Rho – guanine nucleotide exchange factor with which spinophilin interacts ( Ryan et al., 2005 ). "
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    ABSTRACT: The adaptive immune response is initiated by the presentation of peptides bound to major histocompatibility complex molecules on dendritic cells (DCs) to antigen-specific T lymphocytes at a junction termed the immunological synapse. Although much attention has been paid to cytoplasmic events on the T cell side of the synapse, little is known concerning events on the DC side. We have sought signal transduction components of the neuronal synapse that were also expressed by DCs. One such protein is spinophilin, a scaffolding protein of neuronal dendritic spines that regulates synaptic transmission. In inactive, immature DCs, spinophilin is located throughout the cytoplasm but redistributes to the plasma membrane upon stimulus-induced maturation. In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner. It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo. Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.
    The Journal of Cell Biology 05/2008; 181(2):203-11. DOI:10.1083/jcb.200711149 · 9.83 Impact Factor
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