1 / 26

Interaction with the NMDA receptor locks CaMKII in an active conformation

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

hastin
Download Presentation

Interaction with the NMDA receptor locks CaMKII in an active conformation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related