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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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Cdk5 inhibition results in NMDAR-induced PP1 dephosphorylation. (a) Cultured cortical neurons (∼DIV21) were treated with 50 µM roscovitine for different times before proteins were harvested, run on SDS-PAGE, and analyzed by blotting with pT320 and PP1 antibodies. (b) Cultured neurons were infected with recombinant Sindbis viruses encoding GFP, GFP-Cdk5, GFP-P35 or GFP-Cdk5, and GFP-P35 (co-infection). 1 d after infection, the total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, Cdk5, P35, and tubulin (loading control) antibodies. (c) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against Cdk5 (Cdk5 RNAi) or scrambled ShRNA (Con). 5 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and Cdk5 antibodies. Bar graph represents three independent experiments. (d) Cultured cortical neurons were subjected to synaptic (sNMDAR) or extrasynaptic (eNMDAR) NMDA receptor activation (see Materials and methods; same as in Fig. 1 f). Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with p35 and tubulin (loading control) antibodies. (e) Cultured cortical neurons were treated without (Con) or with NMDA (20 or 100 µM) for 10 min with MG132 pre-applied for more than 1 h. Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and P35 antibodies. Bar graph represents three independent experiments. (f) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against p35 (p35 RNAi), scrambled ShRNA (Con), or recombinant lentiviruses encoding p35 RNAi and RNAi-resistant recombinant p35 (p35 RNAi + rescue). 6 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, p35, and tubulin antibodies. Bar graph represents three independent experiments. Solid arrow, RNAi-resistant recombinant p35; hollow arrow, endogenous p35.
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fig5: Cdk5 inhibition results in NMDAR-induced PP1 dephosphorylation. (a) Cultured cortical neurons (∼DIV21) were treated with 50 µM roscovitine for different times before proteins were harvested, run on SDS-PAGE, and analyzed by blotting with pT320 and PP1 antibodies. (b) Cultured neurons were infected with recombinant Sindbis viruses encoding GFP, GFP-Cdk5, GFP-P35 or GFP-Cdk5, and GFP-P35 (co-infection). 1 d after infection, the total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, Cdk5, P35, and tubulin (loading control) antibodies. (c) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against Cdk5 (Cdk5 RNAi) or scrambled ShRNA (Con). 5 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and Cdk5 antibodies. Bar graph represents three independent experiments. (d) Cultured cortical neurons were subjected to synaptic (sNMDAR) or extrasynaptic (eNMDAR) NMDA receptor activation (see Materials and methods; same as in Fig. 1 f). Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with p35 and tubulin (loading control) antibodies. (e) Cultured cortical neurons were treated without (Con) or with NMDA (20 or 100 µM) for 10 min with MG132 pre-applied for more than 1 h. Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and P35 antibodies. Bar graph represents three independent experiments. (f) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against p35 (p35 RNAi), scrambled ShRNA (Con), or recombinant lentiviruses encoding p35 RNAi and RNAi-resistant recombinant p35 (p35 RNAi + rescue). 6 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, p35, and tubulin antibodies. Bar graph represents three independent experiments. Solid arrow, RNAi-resistant recombinant p35; hollow arrow, endogenous p35.

Mentions: We next assessed the mechanism by which PP1 phosphorylation at T320 is regulated in response to synaptic NMDA receptor signaling. Incubation of cortical neurons with the Cdk5 inhibitor roscovitine led to a marked decrease in PP1 phosphorylation at T320 that was maximal after 30–60 min (Fig. 5 a). This is consistent with previous studies that indicated that cyclin-dependent kinases, including Cdk5 in PC12 cells, can phosphorylate PP1 at T320 (Dohadwala et al., 1994; Li et al., 2007). Expression of either Cdk5, or its activator p35, in neurons via recombinant Sindbis virus–mediated infection, also increased PP1 phosphorylation at T320 significantly (Fig. 5 b). In addition, knockdown of endogenous Cdk5 by RNAi resulted in a substantial reduction in PP1 phosphorylation at T320 (Fig. 5 c). Previous studies have indicated that Cdk5 and PP1 can bind to each other (Agarwal-Mawal and Paudel, 2001). We confirmed this interaction in experiments in which Cdk5 was immunoprecipitated (Fig. S4 a). However, NMDA application did not change the amount of co-immunoprecipitated PP1 (Fig. S4 a).


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)

Cdk5 inhibition results in NMDAR-induced PP1 dephosphorylation. (a) Cultured cortical neurons (∼DIV21) were treated with 50 µM roscovitine for different times before proteins were harvested, run on SDS-PAGE, and analyzed by blotting with pT320 and PP1 antibodies. (b) Cultured neurons were infected with recombinant Sindbis viruses encoding GFP, GFP-Cdk5, GFP-P35 or GFP-Cdk5, and GFP-P35 (co-infection). 1 d after infection, the total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, Cdk5, P35, and tubulin (loading control) antibodies. (c) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against Cdk5 (Cdk5 RNAi) or scrambled ShRNA (Con). 5 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and Cdk5 antibodies. Bar graph represents three independent experiments. (d) Cultured cortical neurons were subjected to synaptic (sNMDAR) or extrasynaptic (eNMDAR) NMDA receptor activation (see Materials and methods; same as in Fig. 1 f). Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with p35 and tubulin (loading control) antibodies. (e) Cultured cortical neurons were treated without (Con) or with NMDA (20 or 100 µM) for 10 min with MG132 pre-applied for more than 1 h. Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and P35 antibodies. Bar graph represents three independent experiments. (f) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against p35 (p35 RNAi), scrambled ShRNA (Con), or recombinant lentiviruses encoding p35 RNAi and RNAi-resistant recombinant p35 (p35 RNAi + rescue). 6 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, p35, and tubulin antibodies. Bar graph represents three independent experiments. Solid arrow, RNAi-resistant recombinant p35; hollow arrow, endogenous p35.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Cdk5 inhibition results in NMDAR-induced PP1 dephosphorylation. (a) Cultured cortical neurons (∼DIV21) were treated with 50 µM roscovitine for different times before proteins were harvested, run on SDS-PAGE, and analyzed by blotting with pT320 and PP1 antibodies. (b) Cultured neurons were infected with recombinant Sindbis viruses encoding GFP, GFP-Cdk5, GFP-P35 or GFP-Cdk5, and GFP-P35 (co-infection). 1 d after infection, the total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, Cdk5, P35, and tubulin (loading control) antibodies. (c) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against Cdk5 (Cdk5 RNAi) or scrambled ShRNA (Con). 5 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and Cdk5 antibodies. Bar graph represents three independent experiments. (d) Cultured cortical neurons were subjected to synaptic (sNMDAR) or extrasynaptic (eNMDAR) NMDA receptor activation (see Materials and methods; same as in Fig. 1 f). Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with p35 and tubulin (loading control) antibodies. (e) Cultured cortical neurons were treated without (Con) or with NMDA (20 or 100 µM) for 10 min with MG132 pre-applied for more than 1 h. Total neuronal lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, and P35 antibodies. Bar graph represents three independent experiments. (f) Cultured cortical neurons were infected with recombinant lentiviruses encoding ShRNA against p35 (p35 RNAi), scrambled ShRNA (Con), or recombinant lentiviruses encoding p35 RNAi and RNAi-resistant recombinant p35 (p35 RNAi + rescue). 6 d later, the total lysates were run on SDS-PAGE and analyzed by blotting with pT320, PP1, p35, and tubulin antibodies. Bar graph represents three independent experiments. Solid arrow, RNAi-resistant recombinant p35; hollow arrow, endogenous p35.
Mentions: We next assessed the mechanism by which PP1 phosphorylation at T320 is regulated in response to synaptic NMDA receptor signaling. Incubation of cortical neurons with the Cdk5 inhibitor roscovitine led to a marked decrease in PP1 phosphorylation at T320 that was maximal after 30–60 min (Fig. 5 a). This is consistent with previous studies that indicated that cyclin-dependent kinases, including Cdk5 in PC12 cells, can phosphorylate PP1 at T320 (Dohadwala et al., 1994; Li et al., 2007). Expression of either Cdk5, or its activator p35, in neurons via recombinant Sindbis virus–mediated infection, also increased PP1 phosphorylation at T320 significantly (Fig. 5 b). In addition, knockdown of endogenous Cdk5 by RNAi resulted in a substantial reduction in PP1 phosphorylation at T320 (Fig. 5 c). Previous studies have indicated that Cdk5 and PP1 can bind to each other (Agarwal-Mawal and Paudel, 2001). We confirmed this interaction in experiments in which Cdk5 was immunoprecipitated (Fig. S4 a). However, NMDA application did not change the amount of co-immunoprecipitated PP1 (Fig. S4 a).

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