Limits...
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.

Show MeSH

Related in: MedlinePlus

PP1 dephosphorylation is mediated via auto-dephosphorylation. (a) Cultured cortical neurons were treated without (Con) or with the PP1/PP2A inhibitor OA (200 nM) or the PP2A inhibitor fostriecin (200 nM) for 10 or 30 min. Total cell lysates were run on SDS-PAGE and analyzed by blotting with pT320 or PP1 antibodies. (b) Cultured cortical neurons were treated without (Con) or with NMDA (100 µM) or OA (200 nM) or a combination of OA+NMDA (with OA pre-applied) for 10 min. Total cellular lysates were run on SDS-PAGE and analyzed by blotting with pT320 and PP1 antibodies. Bar graph represents three independent experiments. (c) HEK 293 cells were transfected with PP1α wild type (PP1α WT) or a phosphorylation blocking mutant at T320 (PP1α T320A). Cell lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and myc antibodies. Solid arrows, recombinant PP1; hollow arrows, endogenous PP1. (d) Cultured cortical neurons were infected with recombinant Sindbis virus encoding GFP, PP1α WT-myc-His, or PP1α (T320A) myc-His. 1 d after infection, neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, myc, and tubulin (loading control) antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: PP1 dephosphorylation is mediated via auto-dephosphorylation. (a) Cultured cortical neurons were treated without (Con) or with the PP1/PP2A inhibitor OA (200 nM) or the PP2A inhibitor fostriecin (200 nM) for 10 or 30 min. Total cell lysates were run on SDS-PAGE and analyzed by blotting with pT320 or PP1 antibodies. (b) Cultured cortical neurons were treated without (Con) or with NMDA (100 µM) or OA (200 nM) or a combination of OA+NMDA (with OA pre-applied) for 10 min. Total cellular lysates were run on SDS-PAGE and analyzed by blotting with pT320 and PP1 antibodies. Bar graph represents three independent experiments. (c) HEK 293 cells were transfected with PP1α wild type (PP1α WT) or a phosphorylation blocking mutant at T320 (PP1α T320A). Cell lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and myc antibodies. Solid arrows, recombinant PP1; hollow arrows, endogenous PP1. (d) Cultured cortical neurons were infected with recombinant Sindbis virus encoding GFP, PP1α WT-myc-His, or PP1α (T320A) myc-His. 1 d after infection, neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, myc, and tubulin (loading control) antibodies.

Mentions: Although our results suggested that reduced Cdk5 kinase activity is primarily responsible for the reduced phosphorylation of PP1 observed in response to NMDA signaling, we were also interested in identifying the phosphatase that dephosphorylated PP1 at pT320. Incubation of cortical neurons with OA (200 nM), but not the PP2A-specific inhibitor fostriecin (200 nM), led to an increase in PP1 phosphorylation (Fig. 6 a). Moreover, OA treatment blocked the effect of NMDA application on PP1 dephosphorylation (Fig. 6 b). These results suggest that PP1, or PP1 auto-dephosphorylation, is the phosphatase mechanism responsible for PP1 regulation during basal and NMDA application. In the course of testing the specificity of the pT320 antibody we found that expression of active PP1, i.e., mutant PP1α in which T320 was changed to alanine, led to a decrease in phosphorylation of endogenous PP1 in HEK 293 cells (Fig. 6 c, hollow arrowhead). Expression of recombinant active PP2A (Fig. S5 a) or calcineurin mutants (Fig. S5 b) did not affect phosphorylation of endogenous PP1. To further examine this, we coexpressed PP1α (T320A) in HEK 293 cells with various PP1 isoforms tagged with YFP and examined their phosphorylation level (Fig. S5 c). Expression of PP1α (T320A) substantially reduced phosphorylation of all PP1 isoforms normalized to their respective total proteins. This effect also holds in cortical neurons as expression of PP1α (T320A), but not wild-type PP1, in cortical neurons resulted in reduced phosphorylation of endogenous PP1 (Fig. 6 d). The data suggest, therefore, that PP1 can not only auto-dephosphorylate itself, but also trans-dephosphorylate other PP1 molecules.


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)

PP1 dephosphorylation is mediated via auto-dephosphorylation. (a) Cultured cortical neurons were treated without (Con) or with the PP1/PP2A inhibitor OA (200 nM) or the PP2A inhibitor fostriecin (200 nM) for 10 or 30 min. Total cell lysates were run on SDS-PAGE and analyzed by blotting with pT320 or PP1 antibodies. (b) Cultured cortical neurons were treated without (Con) or with NMDA (100 µM) or OA (200 nM) or a combination of OA+NMDA (with OA pre-applied) for 10 min. Total cellular lysates were run on SDS-PAGE and analyzed by blotting with pT320 and PP1 antibodies. Bar graph represents three independent experiments. (c) HEK 293 cells were transfected with PP1α wild type (PP1α WT) or a phosphorylation blocking mutant at T320 (PP1α T320A). Cell lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and myc antibodies. Solid arrows, recombinant PP1; hollow arrows, endogenous PP1. (d) Cultured cortical neurons were infected with recombinant Sindbis virus encoding GFP, PP1α WT-myc-His, or PP1α (T320A) myc-His. 1 d after infection, neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, myc, and tubulin (loading control) antibodies.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: PP1 dephosphorylation is mediated via auto-dephosphorylation. (a) Cultured cortical neurons were treated without (Con) or with the PP1/PP2A inhibitor OA (200 nM) or the PP2A inhibitor fostriecin (200 nM) for 10 or 30 min. Total cell lysates were run on SDS-PAGE and analyzed by blotting with pT320 or PP1 antibodies. (b) Cultured cortical neurons were treated without (Con) or with NMDA (100 µM) or OA (200 nM) or a combination of OA+NMDA (with OA pre-applied) for 10 min. Total cellular lysates were run on SDS-PAGE and analyzed by blotting with pT320 and PP1 antibodies. Bar graph represents three independent experiments. (c) HEK 293 cells were transfected with PP1α wild type (PP1α WT) or a phosphorylation blocking mutant at T320 (PP1α T320A). Cell lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and myc antibodies. Solid arrows, recombinant PP1; hollow arrows, endogenous PP1. (d) Cultured cortical neurons were infected with recombinant Sindbis virus encoding GFP, PP1α WT-myc-His, or PP1α (T320A) myc-His. 1 d after infection, neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, myc, and tubulin (loading control) antibodies.
Mentions: Although our results suggested that reduced Cdk5 kinase activity is primarily responsible for the reduced phosphorylation of PP1 observed in response to NMDA signaling, we were also interested in identifying the phosphatase that dephosphorylated PP1 at pT320. Incubation of cortical neurons with OA (200 nM), but not the PP2A-specific inhibitor fostriecin (200 nM), led to an increase in PP1 phosphorylation (Fig. 6 a). Moreover, OA treatment blocked the effect of NMDA application on PP1 dephosphorylation (Fig. 6 b). These results suggest that PP1, or PP1 auto-dephosphorylation, is the phosphatase mechanism responsible for PP1 regulation during basal and NMDA application. In the course of testing the specificity of the pT320 antibody we found that expression of active PP1, i.e., mutant PP1α in which T320 was changed to alanine, led to a decrease in phosphorylation of endogenous PP1 in HEK 293 cells (Fig. 6 c, hollow arrowhead). Expression of recombinant active PP2A (Fig. S5 a) or calcineurin mutants (Fig. S5 b) did not affect phosphorylation of endogenous PP1. To further examine this, we coexpressed PP1α (T320A) in HEK 293 cells with various PP1 isoforms tagged with YFP and examined their phosphorylation level (Fig. S5 c). Expression of PP1α (T320A) substantially reduced phosphorylation of all PP1 isoforms normalized to their respective total proteins. This effect also holds in cortical neurons as expression of PP1α (T320A), but not wild-type PP1, in cortical neurons resulted in reduced phosphorylation of endogenous PP1 (Fig. 6 d). The data suggest, therefore, that PP1 can not only auto-dephosphorylate itself, but also trans-dephosphorylate other PP1 molecules.

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