Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization

Watkins, Darryl S.; True, Jason D.; Mosley, Amber L.; Baucum, Anthony J.

doi:10.3390/proteomes6040053

Cited by 13 publications

(15 citation statements)

References 94 publications

(136 reference statements)

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“…To identify signaling nodes that are regulated by fucosylation, we treated 4T1 metastatic mammary tumor cells with the fucosylation inhibitor, 2FF, or DMSO ( Supplementary Figure S1A ) and performed TMT proteomics ( Supplementary Figure S1B ) [ 27 ]. This analysis identified 5750 proteins and quantified 5288 of them by MS2-based TMT.…”

Section: Resultsmentioning

confidence: 99%

NF-κB Signaling Is Regulated by Fucosylation in Metastatic Breast Cancer Cells

et al. 2020

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A growing body of evidence indicates that the levels of fucosylation correlate with breast cancer progression and contribute to metastatic disease. However, very little is known about the signaling and functional outcomes that are driven by fucosylation. We performed a global proteomic analysis of 4T1 metastatic mammary tumor cells in the presence and absence of a fucosylation inhibitor, 2-fluorofucose (2FF). Of significant interest, pathway analysis based on our results revealed a reduction in the NF-κB and TNF signaling pathways, which regulate the inflammatory response. NF-κB is a transcription factor that is pro-tumorigenic and a prime target in human cancer. We validated our results, confirming that treatment of 4T1 cells with 2FF led to a decrease in NF-κB activity through increased IκBα. Based on these observations, we conclude that fucosylation is an important post-translational modification that governs breast cancer cell signaling.

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Section: Resultsmentioning

confidence: 99%

NF-κB Signaling Is Regulated by Fucosylation in Metastatic Breast Cancer Cells

et al. 2020

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“…Mechanistically, how spinophilin decreases surface expression could involve clathrin-mediated endocytosis given that we have previously observed clathrin in spinophilin co-IPs (Watkins et al, 2019) and GluN2B-containing NMDA receptors undergo clathrin-mediated endocytosis (Wu et al, 2017b). Moreover, alterations in vesicle trafficking could involve the motor protein, myosin-Va, since we have previously found that both spinophilin and GluN2B interact with Myosin-Va (Baucum et al, 2013;Hiday et al, 2017;Salek et al, 2019; and others have shown that myosin-Va associates with vesicles and is critical in AMPA receptor trafficking and function (Correia et al, 2008;Rudolf et al, 2011).…”

Section: Discussionmentioning

confidence: 96%

Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function

Salek

Bansal

Berbari

et al. 2021

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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.HIGHLIGHTSSpinophilin bidirectionally regulates GluN2B-containing NMDAR function.Loss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker.Loss of spinophilin in vivo decreases measures of spatial cognitive flexibility.Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks.

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“…The author wishes to make the following corrections to the methods section of their paper [1]: The corrections include: (1) Replace the sentence "Mouse striatum (including both dorsal and ventral (accumbens) striatum or olfactory tubercle) was homogenized and sonicated in 1 mL in a low-ionic strength Tris buffer containing 20 mM Tris, 10 mM DTT, 2 mM 0.5 M EDTA, 10% Triton X-100, 1% protease inhibitor cocktail (Bimake, Houston, TX, USA)" with "Mouse striatum (including both dorsal and ventral (accumbens) striatum or olfactory tubercle) was homogenized and sonicated in 1 mL in a low-ionic strength Tris buffer containing 2 mM Tris-HCl, 1 mM DTT, 2 mM EDTA, 1% Triton X-100, and 1X protease inhibitor cocktail (Bimake, Houston, TX, USA)"; (2) replace the sentence "magnetic separation in an immunoprecipitation wash buffer (50 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5% (v/v) Triton X-100)" with "magnetic separation in an immunoprecipitation wash buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5% (v/v) Triton X-100)".…”

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confidence: 99%