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

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.

CaMKII: Ca2+/calmodulin-dependent protein kinase II

•Autoregulatory :•Thr286-autophosphorylated CaMKII•Thr305/306-autophosphorylated CaMKII•Catalytic site : N-terminal

2002 Biochemical Society

Thr286-autophosphorylated CaMKII

2002 Biochemical Society

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:

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

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

MATERIALS AND METHODS

Proteins Purified

GST Cosedimentation Assays

Kinase Assays

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

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

Kinase Assays

Prepare Non-P or [P-T286] CaMKII

In the 1mM Ca2+-dependent or EGTA(Ca2+-independent), respectively

substrate,syntide-2 initiated by the addition of [γ-32P]ATP

RESULTS

Figure1 A

Non-P [P-T286]

Ca2+/calmodulin-dependent

Sequential-P Basal-P: Basal Ca2+-

independent autophosphorylation [P-T286]

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.

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).

Conclusion:

Wild type CaMKII binding with NR2B >Actinin in Ca2+/calmodulin-dependent-P, but opposite to mutation.

CaMKII bind with Actinin in (TT305/306AA).

Figure1 C Thr305 and Thr306 mutated to Alanine (TT305/306AA).

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 .

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 .

Fig. 3. Ca2+/calmodulin and α-actinin compete for binding to CaMKII [P-T286]:

[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]

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

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

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.

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

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)

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,

DISCUSSION

CaMKII Interaction with NR2B

CaMKII Interaction with Densin

CaMKII Interaction with α-actinin

Implications for CaMKII signaling complexes

CaMKII Interaction with NR2B

Autophosphorylation at Thr286 is also necessary for interaction with a high affinity binding site in NR2B corresponding

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.

CaMKII Interaction with α-actinin Implications for CaMKII signaling

complexes.

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