Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction

Hiday, Andrew C.; Edler, Michael; Salek, Asma B.; Morris, Cameron W.; Thang, Morrent; Rentz, Tyler J.; Rose, Kristie L.; Jones, Lisa M.; Baucum, Anthony J.

doi:10.1155/2017/4153076

Cited by 16 publications

(47 citation statements)

References 60 publications

(93 reference statements)

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“…We measured CK2 activity indirectly by using an antibody against the phospho-CK2 substrate (motif pS/pTDXE) to measure the amount of phosphorylated CK2 substrates. This method is widely used in literature to measure kinase activity, such as activity of the following kinases CK2 [ 16 , 49 , 50 , 51 , 52 ], AMPK [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], AMT/ATR [ 64 , 65 , 66 , 67 , 68 , 69 , 70 ], PKA [ 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 ], and PKC [ 77 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Both CK2 knockdown and CX-4945 treatment inhibited the proliferation of CCA cells.…”

Section: Discussionmentioning

confidence: 99%

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Lertsuwan

Sawasdichai

et al. 2018

Cancers

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Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

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Section: Discussionmentioning

confidence: 99%

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Lertsuwan

Sawasdichai

et al. 2018

Cancers

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“…We created constructs that encoded HA-tagged human spinophilin [62] along with a P2A sequence and a far-red fluorescent protein (mNeptune3 [71]). Mice were generated from these constructs by the Vanderbilt Transgenic Mouse/ESC Shared Resource (see methods).…”

Section: Resultsmentioning

confidence: 99%

“…All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals as disseminated by the U.S. National Institutes of Health and were approved by Indiana University-Purdue University School of Science Animal Care and Use Committees (Approval #SC270R). A human, HA-tagged spinophilin construct [62] containing a P2A sequence and the mNeptune 3 protein were assembled into the pBigT vector between the ClaI and SacI restriction sites (Figure 1A). Gene files were assembled in SnapGene (GSL Biotech, Chicago, IL, USA) or Vector NTI (ThermoFisher Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…DA depletion, which decreases DA terminals in the striatum, modulates spinophilin interactions within the striatum. Specifically, there were increases in spinophilin binding to PP1; however, the interactions of spinophilin with a plurality of spinophilin-associated proteins (SpAPs) were decreased [61,62]. We have shown that whole-body spinophilin knockout (KO) mice do not undergo d-amphetamine-induced behavioral sensitization [63].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants.

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“…Overexpression of the catalytic subunit of PKA (PKAc) in HEK293 cells increases spinophilin and GluN2B interaction PKA phosphorylates both spinophilin and NMDA receptor subunits (Hsieh-Wilson et al 2003;Murphy et al 2014) and our previous studies have found that PKA can regulate spinophilin interactions (Hiday et al 2017). V5-tagged GluN2B Tail and HA-tagged spinophilin were co-expressed in HEK293 cells alone or alongside a Myc-tagged form of the catalytic subunit of protein kinase A (PKAc).…”

Section: Resultsmentioning

confidence: 95%

Spinophilin regulates phosphorylation and interactions of the GluN2B subunit of the N‐methyl‐d‐aspartate receptor

Salek

Edler

McBride

et al. 2019

Journal of Neurochemistry

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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 (PP1) in hippocampal neurons increases NMDAR activity, an effect abrogated by loss of spinophilin, the major PP1‐targeting protein in the postsynaptic density. 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 human embryonic kidney 293 FT 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 postsynaptic density‐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. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/

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Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction

Cited by 16 publications

References 60 publications

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

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

Spinophilin regulates phosphorylation and interactions of the GluN2B subunit of the N‐methyl‐d‐aspartate receptor

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