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Casein Kinase 2 Regulates the NR2 Subunit Composition of Synaptic NMDA Receptor

Antonio Sanz- Clemente , Jose A.Matta , John T.R. Isaac and Katherine W. Roche. National Institute of Neurological Disorders and Stroke, National Institutes of Health , Bethesda, MD 20892, USA. Casein Kinase 2 Regulates the NR2 Subunit Composition of Synaptic NMDA Receptor.

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Casein Kinase 2 Regulates the NR2 Subunit Composition of Synaptic NMDA Receptor

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  1. Antonio Sanz-Clemente, Jose A.Matta, John T.R. Isaac and Katherine W. Roche. National Institute of NeurologicalDisorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA Casein Kinase 2 Regulates the NR2 Subunit Composition of Synaptic NMDA Receptor Bertille LORGEOUX Emilie DESFOSSES M2 Neuropsychopharmacologie

  2. Introduction NR1 • NMDAR • Casein kinase 2 (CK2) : Serine/threonine kinase NR2B CK2 S1480 P α α β β

  3. Whatis the main molecularmecanism for the switchbetween NR2A and NR2B, comprisingsynapticNMDARs?

  4. Material & Method Immunoprecipitation Elution + Centrifugation Centrifugation

  5. Material& Method Immunoblotting Samples Bigproteins Protein of interest Label withspecificantibody and secondaryantibody Protein blot on nitrocellulose Small proteins SDS-PAGE

  6. Test in vitro (1/2) WT (ESDV) S/A (EADV) + CK2 and radioactive ATP +/- TBB for WT NR2 C-term NR2A GST WT (ESDV) S/A (EADV) NR2B

  7. Test in vitro (2/2) • The C-terminal serine isnecessary for the NR2 subunit phosphorylation by CK2 • NR2A isless sensitive than NR2B for the CK2 phosphorylation

  8. Test in vivo (1/2) +/- TBB Lysis and ImmunoprecipitationwithAntibodies for NR2A and NR2 B Phosphorylation Assaywith CK2 Cortical neuron culture

  9. Test In vivo (2/2) • Endogenous NR2B isalsophosphorylated by CK2 • Endogenous NR2A is not efficientlyphosphorylated by CK2

  10. PSD-95 and NR2 subunit interaction (1/2) Cotransfection +/- TBB Lysis and coimmunoprecipitationwith NR2A and NR2B antibodies HEK293 : NR1 + PSD95 + GFP-NR2B +/- TBB Immunoblot figure 1D HEK293 : NR1 + PSD95 + GFP-NR2A

  11. PSD-95 and NR2 subunit interaction (2/2) • The TBB treatement increases the asssociation between PSD95 and NR2B • It is not the case of NR2A which is not affected by the TBB treatement

  12. Effect of inhibiting CK2 activity on the surface expression of the NR2A and NR2B +/- TBB Immunoblotwith NR2A, NR2B, NR1, GluR1 and synaptophysinantibodies Biotinylation and SDS-PAGE Cortical neuron culture

  13. Effect of inhibiting CK2 activity on the surface expression of the NR2A and NR2B  Inhibition of CK2 activityincrease the surface expression of NR2B but not the surface expression of NR2A.

  14. Visualization of the receptor surface expression Immunofluorescence assayprotocol : permeabilization GFP-NR2A GFP-NR2B Anti GFP antibody Surface receptorslabeled in white Internal pool receptorslabeled in green ±TBB

  15. Visualization of the receptor surface expression

  16. Does CK2 regulates the synaptic expression of NR2 subunits?  CK2 regulates the surface expression and the synapticlocalization of NR2 subunits in an inverse manner

  17. Effect of the mutation of E1479 on the synaptic NR2 subunitexpresion  The inhibition of NR2B phosphhorylation on S1480 stabilizes the NR2B expression to the surface

  18. Effect of the E mutation on the interaction between NR2 subunits and PSD95  The non phosphorylation of NR2B on S1480 increase the NR2B interaction with PSD-95 thatsuggesting the phosphorylation of S1480 regulates the synaptic expression of NR2B

  19. Effect of CK2 on NMDAR endocytosis Hippocampalneuronstranfectedwith GFP-NR2B (WT or E1479) : Immunofluorescence assayprotocol : Internal pool receptorslabeled in white Anti GFP antibody permeabilization Surface receptorslabeled in green GFP-NR2B (Wt or E1479) ±TBB E1479 mutation internalization

  20. Effect of CK2 on NMDAR endocytosis  CK2 increaseendocytosis via S1480 phosphorylation NR2B endocytosisisreduce by CK2 inhibition

  21. Does phosphorylation in the PDZ-domain of NR2B regulatesits surface expression and endocytosis via phosphorylation of Y1472? Hippocampalneuronstranfectedwith GFP-NR2B with mutations in the YEKL and/or PDZ-domain : GFP-NR2B (Wt ,E1479Q, S1480E,Y1472A) Internal pool receptorslabeled in white Anti GFP antibody permeabilization Surface receptorslabeled in green E1479 mutation Immunofluorescence assayprotocol : S1480 mutation Y1472 mutation internalization

  22. Does phosphorylation in the PDZ-domain of NR2B regulatesits surface expression and endocytosis via phosphorylation of Y1472? The phosphorylation in the PDZ-domain and in the YEKL endocytic motif, share a commonmolecularmechanism to regulate NR2B endocytosis.

  23. Effect of CK2 inhibition on Y1472 phosphorylation  The reduction of S1480 phosphorylation in presence of TBB isconcomittant to the increase in Y1472 phosphorylation.

  24. Mecanismduring the development : NR2B pS1480 phosphorylation and CK2 activity  The switch NR2B/NR2A thanks to the CK2 occursduring the criticalperiod : P9-P15

  25. CK2 activity in synapticdevelopment  The association of CK2 on SPM at P13 provesthatthis kinase is important in synapticdevelopment

  26. Phosphorylation of S1480 and Y1472 in synapticdevelopment  The phosphorylation « transfert » betwenn S1480 and Y1472 occursalsoduring the criticalperiod : important for the synapticdevelopment

  27. Effect of drugs on NR2B S1480 phosphorylation Phosphorylation on NR2B S1480 isinduce by synapticactivity. TTX, bicuculline or KCl Isolation of cellular membrane Immunoblotwith NR2B or phopho-specific NR2B S1480 antibodies Cortical neuron culture

  28. Effect of drugs on NR2B S1480 phosphorylation  Phosphorylation on NR2B S1480 isinduce by activitythatisdependant of synaptic NMDAR activation. MK-801 or APV Immunoblotwith NR2B or phopho-specific NR2B S1480 antibodies Isolation of cellular membrane Cortical neuron culture

  29. NR2A expression and NR2B S1480 phosphorylation The stimuli required for NMDAR switchisimplicated in NR2B S1480 phosphorylation Immunoblotwith, NR2A, NR2B or phopho-specific NR2B S1480 antibodies Isolation of cortical synaptosomes Cortical synaptosomesfrommice (WT or NR2A KO

  30. Role of CK2 in the swich of NR2 subunitafter induction of LTP + NBQX + Picrotoxin

  31. Role of CK2 in the switch of NR2 subunitsafter an induction of LTP Reductionof sensitivity to infenprodiland speeding of NMDA EPSC kinetics.

  32. Role of CK2 in the switch of NR2 subunitsafter induction of LTP Increasingof sensitivity to infenprodil and no speeding of NMDA EPSC kinetics.

  33. Discussion Model of CK2 regulation of synapticNMDARs

  34. Perspectives Identifying the molecularmechanimsunderlying the regulation of synaptic CK2 activity NR2B S1480 phosphorylation isregulated by synapticactivity and CamKII CamKII phosphorylation of CK2 target CK2 to the synaptic plasma membrane allowing interaction with NR2B. Mechanisms of spatiotemporalregulation of synaptic CK2 activityduring the LTP.

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