Interaction with the NMDA receptor locks CaMKII in an active conformation

1. Interaction with the NMDA receptor locks CaMKII in an active conformation K.-Ulrich Bayer, Paul De Koninck, A. Soren Leonard, Johannes W. Hell & Howard Schulman Group 9 BIPN 148

2. Experiment 1: Hypothesis Is phosphorylation required for the binding of CaMKII to NMDA receptor?

3. Background • 2 sites of interest on NR2B subunit of NMDA receptor: 1. NR2B-C (residues 1,120-1,482) 2. NR2B-P (residues 839-1,120)

4. Methods Showed the binding of CaMKII: -wild type (WT) -T286A (mock P) -T286D (mimic P) with the NR2B-P or NR2B-C domains of NR2B under these conditions: 1. Phosphorylated OR not phosphorylated 2. Ca2+/CaM added OR Ca2+/CaM not added

5. Results • Unphosphorylated WT and T286A did not bind NR2B-P even with Ca/CaM • Autophosphorylated WT binds both sites even after removal of Ca/CaM • AutoP kinase can bind NR2B-C domain even without Ca/CaM • Without autoP the kinase can only bind NR2B-C and only in the presence of Ca/CaM

6. Analysis Ca/Cam alone, without phosphorylation, can induce interaction of CaMKII with NR2B-C

7. Experiment 2 Hypothesis: Does the interaction of CaMKII with NR2B-C increase its affinity for CaM?

8. Methods • Made various NR2B derived peptides • First wash was with IAEDANS-CaM (CaMi), which was a fluorescent tag • Second wash with unlabelled CaM • Measured the rate at which CaMi is replaced

9. Results • N2B-s peptide bound to CaMKII  CaMi remained • N2B-con peptide bound to CaMKII  CaMi dissociated and replaced with unlabelled CaM “N2B-s peptide” is part of NR2B-C peptide “N2B-con peptide” is part of NR2B-P peptide

10. Conclusion • Initial binding of CaMKII to NR2B requires Ca2+/CaM • CaM trapping occurred in N2B-s bound CaMKII • CaM trapping did not occur in N2B-con bound CaMKII

11. Experiment 3

12. Background CaMKII increases its activity if: 1) Ca2+/CaM present 2) autophosphorylation at T286 Experiment 3

13. Hypothesis • Will interaction with NR2B be enough to generate CaMKII activity without autophosphorylation at T286 and without the presence of Ca2+/CaM? Experiment 3

14. Methods • allowed NR2B to bind to the CaMKII by adding Ca2+/CaM • used T286A so that no auto-phosphorylation at the site could occur • added EGTA to remove Ca2+/CaM from the environment • tested to see if there was CaMKII activity Experiment 3

15. Results • “autonomous” refers to the absence of Ca2+/CaM (via EGTA addition) • there was CaMKII with only NR2B added • CaMKII activity occurred WITHOUT the presence of Ca2+/CaM • CaMKII activity occurred WITHOUT the autophosphorylation of T286 Experiment 3

16. Hypothesis • Where on the NR2B peptide does CaMKII interact to generate the autonomous activity? • study the different NR2B-derived peptides on CaMKII activity Experiment 3

17. Methods • used these NR2B-derived peptides: NR2B-C region: N2B-l  residues 1,259-1,310 N2B-s  residues 1,289-1,310 N2B-a  mutated NR2B S1,303A (cannot be phosphorylated) NR2B-P region: N2B-con  residues 1,095-1,119 • added Ca2+ to initiate binding of NR2B peptides with CaMKII • then added EGTA to remove Ca2+/CaM to test CaMKII activity independent of Ca2+/CaM Experiment 3

18. Results • “pre-incubation” of Ca2+ refers to whether or not Ca2+ was supplied in the beginning to allow the N2B-peptide to bind to the CaMKII • interaction of N2B-l with CaMKII increased CaMKII activity the most Experiment 3

19. Analysis • interaction between NR2B and CaMKII is sufficient for CaMKII activity • the region N2B-l interacts with CaMKII to generate CaMKII activity Experiment 3

20. Experiment 4

21. Hypothesis • What is the effect of NR2B binding to CaMKII on the burst phosphorylation of T305/306? Experiment 4

22. Background • burst of phosphorylation at T305/306 inhibits the binding of Ca2+/CaM to CaMKII • burst of phosphorylation at T305/306 can be initiated when CaM dissociates from CaMKII (which occurs when EGTA is added) Experiment 4

23. Methods • used immunoblotting and phospho-specific antibodies to test for burst phosphorylation at T305/306 of CaMKII and for the presence of CaM bound to the CaMKII Experiment 4

24. Results • band shift of phospho-CaMKII refers to the burst phosphorylation at T305/306 • in the NR2B-bound CaMKII where EGTA was added to induce burst phosphorylation at T305/306, no further burst phosphorylation occurred • NR2B-bound CaMKII can suppress the burst phosphorylation of T305/306 of CaMKII by CaM trapping, thereby allowing CaMKII to remain active “soluble” = CaMKII alone “NR2B bound” = NR2B bound CaMKII “ - ” = no EGTA added “ + ” = EGTA added Experiment 4

25. Analysis • NR2B-bound CaMKII can suppress the burst phosphorylation of T305/306 of CaMKII thereby allowing CaMKII to remain active for a longer period of time Experiment 4

26. Conclusion NR2B binding to CaMKII: • results in autonomous CaMKII activity (not dependent on Ca2+/CaM and not dependent on the phosphorylation of T286) • results in CaM trapping which sustains CaMKII activity • suppresses inhibitory phosphorylation of T305/306 in CaMKII