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Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5.

Hou H, Sun L, Siddoway BA, Petralia RS, Yang H, Gu H, Nairn AC, Xia H - J. Cell Biol. (2013)

Bottom Line: The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown.Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons.Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Center, LSU Health Science Center, New Orleans, LA 70112.

ABSTRACT
The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-D-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.

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Endogenous I-2–PP1 complex is regulated by synaptic NMDA receptor signaling. (a) Immunogold localization of I-2 in the CA1 stratum radiatum of the hippocampus from postnatal day 35 (P35) rat. PSD, postsynaptic density; Sp, spine. Bar, 100 nm. (b) Cultured cortical neurons (∼DIV21) were infected with recombinant Sindbis virus encoding CFP–I-2 for 1 d before the neurons were incubated in the absence (Con) or presence of NMDA (20 or 100 µM) for 10 min. Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T72 in CFP–I-2(I-2pT72) or total CFP–I-2 protein (GFP antibody). (c) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Endogenous I-2 protein was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing phospho-T72 of I-2 (I-2pT72) or total I-2 protein (I-2). Bar graph shows data from four experiments. (d) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Proteins were solubilized using RIPA buffer and I-2 was immunoprecipitated followed by blotting for PP1 and T320 phosphorylation. Bar graph shows data from three experiments. (e) Control, synaptic, or extrasynaptic NMDA receptor stimulations were performed on cultured cortical neurons before the proteins were solubilized using RIPA buffer, and I-2 was immunoprecipitated followed by analysis by blotting for PP1. Bar graph shows data from three experiments. (f) Cultured cortical neurons were infected with recombinant Sindbis virus encoding CFP–I-2 (labeled as I-2(WT)) or CFP–2(T72A) (labeled as I-2(T72A)) for 1 d before the RIPA soluble fractions were used for immunoprecipitation with GFP antibody (GFP IP). Samples were analyzed by blotting for GFP and I-2 phosphorylation status on T72. Note: two different exposures of PP1 and I-2pT72 blots are presented.
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fig3: Endogenous I-2–PP1 complex is regulated by synaptic NMDA receptor signaling. (a) Immunogold localization of I-2 in the CA1 stratum radiatum of the hippocampus from postnatal day 35 (P35) rat. PSD, postsynaptic density; Sp, spine. Bar, 100 nm. (b) Cultured cortical neurons (∼DIV21) were infected with recombinant Sindbis virus encoding CFP–I-2 for 1 d before the neurons were incubated in the absence (Con) or presence of NMDA (20 or 100 µM) for 10 min. Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T72 in CFP–I-2(I-2pT72) or total CFP–I-2 protein (GFP antibody). (c) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Endogenous I-2 protein was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing phospho-T72 of I-2 (I-2pT72) or total I-2 protein (I-2). Bar graph shows data from four experiments. (d) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Proteins were solubilized using RIPA buffer and I-2 was immunoprecipitated followed by blotting for PP1 and T320 phosphorylation. Bar graph shows data from three experiments. (e) Control, synaptic, or extrasynaptic NMDA receptor stimulations were performed on cultured cortical neurons before the proteins were solubilized using RIPA buffer, and I-2 was immunoprecipitated followed by analysis by blotting for PP1. Bar graph shows data from three experiments. (f) Cultured cortical neurons were infected with recombinant Sindbis virus encoding CFP–I-2 (labeled as I-2(WT)) or CFP–2(T72A) (labeled as I-2(T72A)) for 1 d before the RIPA soluble fractions were used for immunoprecipitation with GFP antibody (GFP IP). Samples were analyzed by blotting for GFP and I-2 phosphorylation status on T72. Note: two different exposures of PP1 and I-2pT72 blots are presented.

Mentions: In addition to I-1, PP1 is regulated by inhibitor-2 (I-2; Cohen, 1989). I-2 is expressed at high levels in the brain, and has a wide expression in the central nervous system based on in situ studies (Sakagami and Kondo, 1995). However, little is known about I-2 function in neurons. Notably, immunogold electron micrograph (EM) data indicated that I-2 is localized in dendritic spines in hippocampal pyramidal neurons (Fig. 3 a), raising the possibility that it could be involved in regulation of PP1 at synapses.


Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5.

Hou H, Sun L, Siddoway BA, Petralia RS, Yang H, Gu H, Nairn AC, Xia H - J. Cell Biol. (2013)

Endogenous I-2–PP1 complex is regulated by synaptic NMDA receptor signaling. (a) Immunogold localization of I-2 in the CA1 stratum radiatum of the hippocampus from postnatal day 35 (P35) rat. PSD, postsynaptic density; Sp, spine. Bar, 100 nm. (b) Cultured cortical neurons (∼DIV21) were infected with recombinant Sindbis virus encoding CFP–I-2 for 1 d before the neurons were incubated in the absence (Con) or presence of NMDA (20 or 100 µM) for 10 min. Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T72 in CFP–I-2(I-2pT72) or total CFP–I-2 protein (GFP antibody). (c) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Endogenous I-2 protein was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing phospho-T72 of I-2 (I-2pT72) or total I-2 protein (I-2). Bar graph shows data from four experiments. (d) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Proteins were solubilized using RIPA buffer and I-2 was immunoprecipitated followed by blotting for PP1 and T320 phosphorylation. Bar graph shows data from three experiments. (e) Control, synaptic, or extrasynaptic NMDA receptor stimulations were performed on cultured cortical neurons before the proteins were solubilized using RIPA buffer, and I-2 was immunoprecipitated followed by analysis by blotting for PP1. Bar graph shows data from three experiments. (f) Cultured cortical neurons were infected with recombinant Sindbis virus encoding CFP–I-2 (labeled as I-2(WT)) or CFP–2(T72A) (labeled as I-2(T72A)) for 1 d before the RIPA soluble fractions were used for immunoprecipitation with GFP antibody (GFP IP). Samples were analyzed by blotting for GFP and I-2 phosphorylation status on T72. Note: two different exposures of PP1 and I-2pT72 blots are presented.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3824016&req=5

fig3: Endogenous I-2–PP1 complex is regulated by synaptic NMDA receptor signaling. (a) Immunogold localization of I-2 in the CA1 stratum radiatum of the hippocampus from postnatal day 35 (P35) rat. PSD, postsynaptic density; Sp, spine. Bar, 100 nm. (b) Cultured cortical neurons (∼DIV21) were infected with recombinant Sindbis virus encoding CFP–I-2 for 1 d before the neurons were incubated in the absence (Con) or presence of NMDA (20 or 100 µM) for 10 min. Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T72 in CFP–I-2(I-2pT72) or total CFP–I-2 protein (GFP antibody). (c) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Endogenous I-2 protein was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing phospho-T72 of I-2 (I-2pT72) or total I-2 protein (I-2). Bar graph shows data from four experiments. (d) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Proteins were solubilized using RIPA buffer and I-2 was immunoprecipitated followed by blotting for PP1 and T320 phosphorylation. Bar graph shows data from three experiments. (e) Control, synaptic, or extrasynaptic NMDA receptor stimulations were performed on cultured cortical neurons before the proteins were solubilized using RIPA buffer, and I-2 was immunoprecipitated followed by analysis by blotting for PP1. Bar graph shows data from three experiments. (f) Cultured cortical neurons were infected with recombinant Sindbis virus encoding CFP–I-2 (labeled as I-2(WT)) or CFP–2(T72A) (labeled as I-2(T72A)) for 1 d before the RIPA soluble fractions were used for immunoprecipitation with GFP antibody (GFP IP). Samples were analyzed by blotting for GFP and I-2 phosphorylation status on T72. Note: two different exposures of PP1 and I-2pT72 blots are presented.
Mentions: In addition to I-1, PP1 is regulated by inhibitor-2 (I-2; Cohen, 1989). I-2 is expressed at high levels in the brain, and has a wide expression in the central nervous system based on in situ studies (Sakagami and Kondo, 1995). However, little is known about I-2 function in neurons. Notably, immunogold electron micrograph (EM) data indicated that I-2 is localized in dendritic spines in hippocampal pyramidal neurons (Fig. 3 a), raising the possibility that it could be involved in regulation of PP1 at synapses.

Bottom Line: The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown.Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons.Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neuroscience Center, LSU Health Science Center, New Orleans, LA 70112.

ABSTRACT
The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-D-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.

Show MeSH
Related in: MedlinePlus