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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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Synaptic, but not extrasynaptic NMDA receptor stimulation, mediates protein phosphatase 1 activation. (a) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM for 10 min) without or with 100 µM D-APV or 20 µM CNQX (both pre-applied to cultures for 10 min). Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T320 in PP1 (pT320) or total PP1. Bar graph shows data from three experiments. (b) Cultured cortical neurons were incubated in the absence or presence of NMDA (20 and 100 µM for 10 min). Cells were lysed and PP1 activity measured. (c) Hippocampal slices were incubated with NMDA (100 µM for 10 min) and pT320 and total PP1 assayed by immunoblotting as in panel a. (d) Cortical cultures were incubated in the absence or presence of NMDA (20 or 100 µM for 10 min). Cells were lysed and nuclear and cytosolic fractions were prepared. pT320 and total PP1 were assayed by immunoblotting as in panel a. HDAC1 (as a nuclear marker) and I-1 (as a cytosolic marker) were also analyzed by immunoblotting. (e) Cortical cultures were incubated in the absence and presence of NMDA with or without the addition of 2.5 mM Ca2+ to the ACSF. (f) Cortical cultures were subjected to synaptic or extrasynaptic stimulations. Neurons were incubated in the absence or presence of various drugs (APV, bicuculline [BIC], 4AP, MK801, or NMDA): three synaptic NMDA receptor stimulation methods (APV removal, BIC/4-AP, and sNMDAR Stim) were used (see Materials and methods for details). BIC/4AP/MK801: MK801 was added for 5 min after 10 min BIC/4AP application; this protocol thus inactivates synaptic NMDAR signaling through irreversible blockade of the NMDA receptor channel pore with MK801. BIC/4AP/MK801+NMDA: NMDA was applied immediately after MK801 washout following the BIC/4AP/MK801 protocol. This protocol stimulates extrasynaptic NMDA receptors. sNMDAR Stim: co-application of BIC, glycine, and nifedipine, a synaptic NMDA receptor stimulation protocol. eNMDAR20 (100) Stim means that after synaptic stimulation, MK801 is washed in (5 min) and washed out before 20 (100) μM NMDA was applied to specifically activate extrasynaptic NMDA receptors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the control.
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fig1: Synaptic, but not extrasynaptic NMDA receptor stimulation, mediates protein phosphatase 1 activation. (a) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM for 10 min) without or with 100 µM D-APV or 20 µM CNQX (both pre-applied to cultures for 10 min). Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T320 in PP1 (pT320) or total PP1. Bar graph shows data from three experiments. (b) Cultured cortical neurons were incubated in the absence or presence of NMDA (20 and 100 µM for 10 min). Cells were lysed and PP1 activity measured. (c) Hippocampal slices were incubated with NMDA (100 µM for 10 min) and pT320 and total PP1 assayed by immunoblotting as in panel a. (d) Cortical cultures were incubated in the absence or presence of NMDA (20 or 100 µM for 10 min). Cells were lysed and nuclear and cytosolic fractions were prepared. pT320 and total PP1 were assayed by immunoblotting as in panel a. HDAC1 (as a nuclear marker) and I-1 (as a cytosolic marker) were also analyzed by immunoblotting. (e) Cortical cultures were incubated in the absence and presence of NMDA with or without the addition of 2.5 mM Ca2+ to the ACSF. (f) Cortical cultures were subjected to synaptic or extrasynaptic stimulations. Neurons were incubated in the absence or presence of various drugs (APV, bicuculline [BIC], 4AP, MK801, or NMDA): three synaptic NMDA receptor stimulation methods (APV removal, BIC/4-AP, and sNMDAR Stim) were used (see Materials and methods for details). BIC/4AP/MK801: MK801 was added for 5 min after 10 min BIC/4AP application; this protocol thus inactivates synaptic NMDAR signaling through irreversible blockade of the NMDA receptor channel pore with MK801. BIC/4AP/MK801+NMDA: NMDA was applied immediately after MK801 washout following the BIC/4AP/MK801 protocol. This protocol stimulates extrasynaptic NMDA receptors. sNMDAR Stim: co-application of BIC, glycine, and nifedipine, a synaptic NMDA receptor stimulation protocol. eNMDAR20 (100) Stim means that after synaptic stimulation, MK801 is washed in (5 min) and washed out before 20 (100) μM NMDA was applied to specifically activate extrasynaptic NMDA receptors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the control.

Mentions: Previous studies have indicated that PP1 is inhibited by phosphorylation on a conserved threonine in the C-terminal tail (Dohadwala et al., 1994). We initially confirmed that a phospho-antibody raised against phospho-T320 in the PP1α isoform (termed pT320 in this paper) was specific and recognized all four PP1 isoforms expressed in HEK293 cells (Fig. S1, a–c). Bath application of NMDA to primary cortical neurons resulted in a marked decrease in PP1 phosphorylation at T320 (Fig. 1 a; Fig. S1 d). Dephosphorylation of PP1 at T320 was closely correlated with NMDA dosage and application duration, with a maximal effect being obtained with >50 µM NMDA and a significant decrease being observed after 1 min (Fig. S1 d). This bath NMDA application is a standard chemical LTD stimulus. However, treatment with glycine alone, which is used as a chemical LTP stimulus (Lu et al., 2001), had no effect on the level of PP1 phosphorylation at T320 (Fig. S1 e). The bath NMDA effect on PP1 dephosphorylation at T320 was correlated with increased PP1 activity (Fig. 1 b), consistent with the established concept of pT320 being an inhibitory phosphorylation. PP1 dephosphorylation at T320 also occurred in brain hippocampal slices, in response to NMDA application (Fig. 1 c). Notably, bath NMDA application to cortical neurons resulted in dephosphorylation of both cytosolic and nuclear pools of PP1 (Fig. 1 d). Pharmacological experiments indicated that the effect of NMDA on PP1 phosphorylation at T320 was blocked by the NMDA receptor antagonist D-APV, but was not affected by the AMPA receptor antagonist CNQX (Fig. 1 a). Incubation of cortical neurons in calcium-free artificial cerebral spinal fluid (ACSF) blocked the effect of NMDA (Fig. 1 e), supporting the conclusion that calcium influx through NMDA receptors is critical for mediating PP1 dephosphorylation at T320.


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)

Synaptic, but not extrasynaptic NMDA receptor stimulation, mediates protein phosphatase 1 activation. (a) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM for 10 min) without or with 100 µM D-APV or 20 µM CNQX (both pre-applied to cultures for 10 min). Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T320 in PP1 (pT320) or total PP1. Bar graph shows data from three experiments. (b) Cultured cortical neurons were incubated in the absence or presence of NMDA (20 and 100 µM for 10 min). Cells were lysed and PP1 activity measured. (c) Hippocampal slices were incubated with NMDA (100 µM for 10 min) and pT320 and total PP1 assayed by immunoblotting as in panel a. (d) Cortical cultures were incubated in the absence or presence of NMDA (20 or 100 µM for 10 min). Cells were lysed and nuclear and cytosolic fractions were prepared. pT320 and total PP1 were assayed by immunoblotting as in panel a. HDAC1 (as a nuclear marker) and I-1 (as a cytosolic marker) were also analyzed by immunoblotting. (e) Cortical cultures were incubated in the absence and presence of NMDA with or without the addition of 2.5 mM Ca2+ to the ACSF. (f) Cortical cultures were subjected to synaptic or extrasynaptic stimulations. Neurons were incubated in the absence or presence of various drugs (APV, bicuculline [BIC], 4AP, MK801, or NMDA): three synaptic NMDA receptor stimulation methods (APV removal, BIC/4-AP, and sNMDAR Stim) were used (see Materials and methods for details). BIC/4AP/MK801: MK801 was added for 5 min after 10 min BIC/4AP application; this protocol thus inactivates synaptic NMDAR signaling through irreversible blockade of the NMDA receptor channel pore with MK801. BIC/4AP/MK801+NMDA: NMDA was applied immediately after MK801 washout following the BIC/4AP/MK801 protocol. This protocol stimulates extrasynaptic NMDA receptors. sNMDAR Stim: co-application of BIC, glycine, and nifedipine, a synaptic NMDA receptor stimulation protocol. eNMDAR20 (100) Stim means that after synaptic stimulation, MK801 is washed in (5 min) and washed out before 20 (100) μM NMDA was applied to specifically activate extrasynaptic NMDA receptors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the control.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: Synaptic, but not extrasynaptic NMDA receptor stimulation, mediates protein phosphatase 1 activation. (a) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM for 10 min) without or with 100 µM D-APV or 20 µM CNQX (both pre-applied to cultures for 10 min). Proteins were analyzed by SDS-PAGE and immunoblotting with antibody to phospho-T320 in PP1 (pT320) or total PP1. Bar graph shows data from three experiments. (b) Cultured cortical neurons were incubated in the absence or presence of NMDA (20 and 100 µM for 10 min). Cells were lysed and PP1 activity measured. (c) Hippocampal slices were incubated with NMDA (100 µM for 10 min) and pT320 and total PP1 assayed by immunoblotting as in panel a. (d) Cortical cultures were incubated in the absence or presence of NMDA (20 or 100 µM for 10 min). Cells were lysed and nuclear and cytosolic fractions were prepared. pT320 and total PP1 were assayed by immunoblotting as in panel a. HDAC1 (as a nuclear marker) and I-1 (as a cytosolic marker) were also analyzed by immunoblotting. (e) Cortical cultures were incubated in the absence and presence of NMDA with or without the addition of 2.5 mM Ca2+ to the ACSF. (f) Cortical cultures were subjected to synaptic or extrasynaptic stimulations. Neurons were incubated in the absence or presence of various drugs (APV, bicuculline [BIC], 4AP, MK801, or NMDA): three synaptic NMDA receptor stimulation methods (APV removal, BIC/4-AP, and sNMDAR Stim) were used (see Materials and methods for details). BIC/4AP/MK801: MK801 was added for 5 min after 10 min BIC/4AP application; this protocol thus inactivates synaptic NMDAR signaling through irreversible blockade of the NMDA receptor channel pore with MK801. BIC/4AP/MK801+NMDA: NMDA was applied immediately after MK801 washout following the BIC/4AP/MK801 protocol. This protocol stimulates extrasynaptic NMDA receptors. sNMDAR Stim: co-application of BIC, glycine, and nifedipine, a synaptic NMDA receptor stimulation protocol. eNMDAR20 (100) Stim means that after synaptic stimulation, MK801 is washed in (5 min) and washed out before 20 (100) μM NMDA was applied to specifically activate extrasynaptic NMDA receptors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the control.
Mentions: Previous studies have indicated that PP1 is inhibited by phosphorylation on a conserved threonine in the C-terminal tail (Dohadwala et al., 1994). We initially confirmed that a phospho-antibody raised against phospho-T320 in the PP1α isoform (termed pT320 in this paper) was specific and recognized all four PP1 isoforms expressed in HEK293 cells (Fig. S1, a–c). Bath application of NMDA to primary cortical neurons resulted in a marked decrease in PP1 phosphorylation at T320 (Fig. 1 a; Fig. S1 d). Dephosphorylation of PP1 at T320 was closely correlated with NMDA dosage and application duration, with a maximal effect being obtained with >50 µM NMDA and a significant decrease being observed after 1 min (Fig. S1 d). This bath NMDA application is a standard chemical LTD stimulus. However, treatment with glycine alone, which is used as a chemical LTP stimulus (Lu et al., 2001), had no effect on the level of PP1 phosphorylation at T320 (Fig. S1 e). The bath NMDA effect on PP1 dephosphorylation at T320 was correlated with increased PP1 activity (Fig. 1 b), consistent with the established concept of pT320 being an inhibitory phosphorylation. PP1 dephosphorylation at T320 also occurred in brain hippocampal slices, in response to NMDA application (Fig. 1 c). Notably, bath NMDA application to cortical neurons resulted in dephosphorylation of both cytosolic and nuclear pools of PP1 (Fig. 1 d). Pharmacological experiments indicated that the effect of NMDA on PP1 phosphorylation at T320 was blocked by the NMDA receptor antagonist D-APV, but was not affected by the AMPA receptor antagonist CNQX (Fig. 1 a). Incubation of cortical neurons in calcium-free artificial cerebral spinal fluid (ACSF) blocked the effect of NMDA (Fig. 1 e), supporting the conclusion that calcium influx through NMDA receptors is critical for mediating PP1 dephosphorylation at T320.

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