1. Differential modulation of Ca 2+ /calmodulin- dependent protein kinase II (CaMKII) activity by regulated interactions with NMDA receptor NR2B subunits and α-Actinin. A.J. Robison et al., JBC Papers Published on September 19, 2005 銘傳大學生科四甲 學生 : 林志隆 報告日期 :2005.10.11

2. Introduction CaMKII: Ca 2+ /calmodulin-dependent protein kinase II. CaMKAPs :Neuronal Ca 2+ /calmodulin- dependent protein kinase II (CaMKII) interacts with several CaMKII Associated Proteins (CaMKAPs)/NR2B, densin-180,α-Actinin.

3. CaMKII: Ca 2+ /calmodulin-dependent protein kinase II Autoregulatory : Thr 286 -autophosphorylated CaMKII Thr 305/306 -autophosphorylated CaMKII Catalytic site : N-terminal 2002 Biochemical Society

4. Thr 286 -autophosphorylated CaMKII 2002 Biochemical Society

5. NR2B:N-methyl-D-aspartate (NMDA) receptor of subunits /NR1, NR2A–D, NR3A–B.(Strack et al.,1998)(Leonard et al.,1999)(Gardoni et al.,1999) Densin-180:(leucine-rich repeat) transmembrane glycoprotein (Walikonis et al.,2001) α-Actinin: F-actin-binding protein (Dhavan et al.,2002) CaMKAPs:

6. NATURE REVIEWS | NEUROSCIENCE OCTOBER 2004 www.nature.com/reviews/neuro

7. J.M. Loftis, A. Janowsky / Pharmacology & Therapeutics 97 (2003) 55–85

8. MATERIALS AND METHODS Proteins Purified GST Cosedimentation Assays Kinase Assays

9. Proteins Purified Purified glutathione-S-transferase (GST) fusion proteins containing CaMKAP fragments. Glutathione-S-transferases (GSTs) are important in the detoxification by conjugating the thiol group. GST-NR2B contains amino acids 1260-1339 of NR2B GST-densin contains amino acids 1247-1542 of densin GST-actinin contains amino acids 819-894 of α-actinin

10. GST Cosedimentation Assays Purified GST fusion proteins and CaMKII and glutathione agarose beads in pulldown (PD) buffer Beads were sedimented by centrifugation and washed in PD buffer SDS-PAGE,Ponceau S (dye) and quantified

11. Kinase Assays Prepare Non-P or [P-T 286 ] CaMKII In the 1mM Ca 2+ - dependent or EGTA(Ca 2+ -independent), respectively substrate,syntide-2 initiated by the addition of [γ- 32 P]ATP

12. RESULTS Figure1 A Non-P [P-T 286 ] Ca 2+ /calmodulin- dependent Sequential-P Basal-P: Basal Ca 2+ - independent autophosphorylation [P-T 286 ]

13. Conclusion: No binding with NR2B in Non-P and Basal-P. reduced level to bind densin (33±5%) in Non-P. Figure1 A Regulation of CaMKII binding to CaMKAPs by autophosphorylation.

14. Figure1 B Regulation of CaMKII binding to CaMKAPs by autophosphorylation. compare white and black bars. reduced level to bind densin and NR2B. ( * : p< 0.01 vs. Ca 2+ /CaM-Dep.  : p< 0.001 vs. Non-P. †: not significantly greater that the non-specific binding to GST alone).

15. Conclusion: Wild type CaMKII binding with NR2B >Actinin in Ca 2+ /calmodulin- dependent-P, but opposite to mutation. CaMKII bind with Actinin in (TT 305/306 AA). Figure1 C Thr 305 and Thr 306 mutated to Alanine (TT 305/306 AA).

16. Regulation of CaMKII binding to CaMKAPs by Ca 2+ /calmodulin. Figure 2 Conclusion: Ca 2+ had no significant effect on the binding of non-phosphorylated CaMKII. A little decrease in Ca 2+ /calmodulin- dependent. CaMKII not binding with Actinin in in Ca 2+ /calmodulin- dependent P-T 286.

17. Regulation of CaMKII binding to CaMKAPs by Ca 2+ /calmodulin. Figure 2 Conclusion: Ca 2+ had no significant effect on the binding of non-phosphorylated CaMKII. CaMKII not binding with Actinin in in Ca 2+ /calmodulin- dependent P-T 286.

18. Fig. 3. Ca 2+ /calmodulin and α-actinin compete for binding to CaMKII [P-T 286 ]: [P-T 286 ] Conclusion: The affinity of CaMKII for calmodulin is increased by Thr 286 autophosphorylat ion Presumably sufficient to allow Ca 2+ /calmodulin to displace α-actinin from [P-T 286 ]CaMKII [P-T 286 ]

19. Fig 3B. To determine whether calmodulin and actinin also compete for nonphosphorylated CaMKII Ca2+/calmodulin-dependent CaMKII activity was assayed using the indicated calmodulin concentrations in the presence ( ) or absence ( ) of 1 µM His6-actinin. Conclusion: actinin and calmodulin compete for binding to both [P- T 286 ]CaMKII and Non-P CaMKII. actinin no actinin

20. Fig4 A CaMKAPs regulate CaMKII activity. Conclusion: no effect on the Ca 2+ - independent activity of [P- T 286 ]CaMKII actinin inhibited Ca 2+ /calmodulin- dependent substrate (autocamtide-2) phosphorylation

21. Fig4 B NR2B inhibition of CaMKII Conclusion: NR2B potently inhibited both Ca 2+ /calmodulin- dependent CaMKII autophosphorylation NR2B inhibited the Ca 2+ - independent activity of [P- T 286 ] CaMKII.

22. Fig 5 Kinetics of CaMKII Inhibition by NR2B. Fig A: using varying syntide-2 concentrations and constant ATP (saturating). conclusion: Inhibition by NR2B was noncompetitive with syntide-2. Fig B: varying ATP concentrations and constant syntide-2. conclusion: inhibition was uncompetitive with variable ATP concentrations (parallel). substrate :syntide-2

23. Fig 5 Kinetics of CaMKII Inhibition by NR2B. Conclusion: Fig C: inhibition was competitive with variable AC2 Fig D: inhibition was uncompetitive with variable ATP concentrations substrate :autocamtide- 2 (AC2)

24. Fig6 Nucleotides stabilize Ca 2+ -dependent binding of CaMKII to NR2B. Conclusion: Nonphosphorylated CaMKII binds GST-NR2B in a Ca 2+ concentration- dependent manner in the presence of ADP. Not bind NR2B in the absence of ADP (right). with or without 100 μM ADP,

25. DISCUSSION CaMKII Interaction with NR2B CaMKII Interaction with Densin CaMKII Interaction with α-actinin Implications for CaMKII signaling complexes

26. CaMKII Interaction with NR2B Autophosphorylation at Thr 286 is also necessary for interaction with a high affinity binding site in NR2B corresponding

27. CaMKII Interaction with Densin CaMKII binding to densin is unaffected by Ca 2+ /calmodulin binding or by Ca 2+ - independent autophosphorylation at Thr 305/306 and other sites.

28. CaMKII Interaction with α-actinin Implications for CaMKII signaling complexes. THANK YOU