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

1. Differential modulation of Ca2+/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: Ca2+/calmodulin-dependent protein kinase II. • CaMKAPs :Neuronal Ca2+/calmodulin-dependent protein kinase II (CaMKII) interacts with several CaMKII Associated Proteins (CaMKAPs)/NR2B, densin-180,α-Actinin.

3. CaMKII: Ca2+/calmodulin-dependent protein kinase II 2002 Biochemical Society • Autoregulatory : • Thr286-autophosphorylated CaMKII • Thr305/306-autophosphorylated CaMKII • Catalytic site : N-terminal

4. Thr286-autophosphorylated CaMKII 2002 Biochemical Society

5. CaMKAPs: • 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)

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

11. Kinase Assays

12. RESULTS • Figure1 A • Non-P • [P-T286] Ca2+/calmodulin-dependent • Sequential-P • Basal-P: Basal Ca2+-independent autophosphorylation [P-T286]

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

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. Ca2+/CaM-Dep. : p< 0.001 vs. Non-P. †: not significantly greater that the non-specific binding to GST alone).

15. Figure1 C Thr305 and Thr306 mutated to Alanine (TT305/306AA). • Conclusion: • Wild type CaMKII binding with NR2B >Actinin in Ca2+/calmodulin-dependent-P, but opposite to mutation. • CaMKII bind with Actinin in (TT305/306AA).

16. Regulation of CaMKII binding to CaMKAPs by Ca2+/calmodulin. Figure 2 • Conclusion: • Ca2+ had no significant effect on the binding of non-phosphorylated CaMKII. A little decrease in Ca2+/calmodulin-dependent. • CaMKII not binding with Actinin in in Ca2+/calmodulin-dependent P-T286 .

17. Regulation of CaMKII binding to CaMKAPs by Ca2+/calmodulin. Figure 2 • Conclusion: • Ca2+ had no significant effect on the binding of non-phosphorylated CaMKII. • CaMKII not binding with Actinin in in Ca2+/calmodulin-dependent P-T286 .

18. Fig. 3. Ca2+/calmodulin and α-actinin compete for binding to CaMKII [P-T286]: • Conclusion: • The affinity of CaMKII for calmodulin is increased by Thr286autophosphorylation • Presumably sufficient to allow Ca2+/calmodulin to displace α-actinin from [P-T286]CaMKII [P-T286] [P-T286]

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-T286]CaMKII and Non-P CaMKII. actinin no actinin

20. Fig4 A CaMKAPs regulate CaMKII activity. • Conclusion: • no effect on the Ca2+-independent activity of [P-T286]CaMKII • actinin inhibited Ca2+/calmodulin-dependent substrate (autocamtide-2) phosphorylation

21. Fig4 B NR2B inhibition of CaMKII • Conclusion: • NR2B potently inhibited both Ca2+/calmodulin-dependent CaMKII autophosphorylation • NR2B inhibited the Ca2+-independent activity of [P-T286] CaMKII.

22. Fig 5 Kinetics of CaMKII Inhibition by NR2B. substrate :syntide-2 • 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).

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 Ca2+-dependent binding of CaMKII to NR2B. • Conclusion: • Nonphosphorylated CaMKII binds GST-NR2B in a Ca2+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 Thr286 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 Ca2+/calmodulin binding or by Ca2+-independent autophosphorylation at Thr305/306 and other sites.

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