Transcript CaMKII

Differential modulation of Ca2+/calmodulindependent 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+/calmodulindependent protein kinase II (CaMKII)
interacts with several CaMKII
Associated Proteins (CaMKAPs)/NR2B,
densin-180,α-Actinin.
CaMKII: Ca2+/calmodulin-dependent
protein kinase II
2002 Biochemical Society
•Autoregulatory :
•Thr286-autophosphorylated CaMKII
•Thr305/306-autophosphorylated CaMKII
•Catalytic site : N-terminal
Thr286-autophosphorylated
CaMKII
2002 Biochemical Society
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)
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
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GST Cosedimentation Assays
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Kinase Assays
Proteins Purified
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Purified glutathione-S-transferase (GST)
fusion proteins containing CaMKAP
fragments.
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Glutathione-S-transferases (GSTs) are
important in the detoxification by conjugating
the thiol group .
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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
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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
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Figure1 A
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Non-P
[P-T286]
Ca2+/calmodulindependent
Sequential-P
Basal-P: Basal Ca2+independent
autophosphorylation
[P-T286]
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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.
Figure1 B Regulation of CaMKII binding to CaMKAPs by
autophosphorylation.

compare white and
black bars.
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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).
Figure1 C
Thr305 and Thr306 mutated to Alanine (TT305/306AA).
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Conclusion:
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Wild type CaMKII binding
with NR2B >Actinin in
Ca2+/calmodulindependent-P, but
opposite to mutation.

CaMKII bind with Actinin
in (TT305/306AA).
Regulation of CaMKII binding to CaMKAPs
by Ca2+/calmodulin. Figure 2
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Conclusion:
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Ca2+ had no significant
effect on the binding of
non-phosphorylated
CaMKII. A little decrease
in Ca2+/calmodulindependent.
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CaMKII not binding with
Actinin in in
Ca2+/calmodulindependent P-T286 .
Regulation of CaMKII binding to
CaMKAPs by Ca2+/calmodulin. Figure 2
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Conclusion:
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Ca2+ had no significant
effect on the binding of
non-phosphorylated
CaMKII.
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CaMKII not binding with
Actinin in in
Ca2+/calmodulindependent P-T286 .
Fig. 3. Ca2+/calmodulin and α-actinin compete for
binding to CaMKII [P-T286]:
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
[P-T286]
[P-T286]
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Conclusion:
The affinity of CaMKII
for calmodulin is
increased by
Thr286autophosphorylat
ion
Presumably sufficient
to allow
Ca2+/calmodulin to
displace α-actinin from
[P-T286]CaMKII
Fig 3B. To determine whether calmodulin and actinin also
compete for nonphosphorylated CaMKII
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Ca2+/calmodulin-dependent
CaMKII activity was assayed
using the indicated calmodulin
concentrations in the presence
( ) or absence ( ) of 1 µM
His6-actinin.
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Conclusion:
actinin and
calmodulin compete
for binding to both [PT286]CaMKII and
Non-P CaMKII.
actinin
no actinin
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Fig4 A CaMKAPs regulate CaMKII activity.
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Conclusion:
no effect on the Ca2+independent activity of [PT286]CaMKII
actinin inhibited
Ca2+/calmodulindependent substrate
(autocamtide-2)
phosphorylation
Fig4 B NR2B inhibition
of CaMKII
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Conclusion:
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NR2B potently inhibited
both Ca2+/calmodulindependent CaMKII
autophosphorylation
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NR2B inhibited the Ca2+independent activity of [PT286] CaMKII.
Fig 5 Kinetics of CaMKII Inhibition by NR2B.
substrate :syntide-2
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Fig A: using varying syntide-2
concentrations and constant
ATP (saturating).
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conclusion:
Inhibition by NR2B was
noncompetitive with syntide-2.
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Fig B: varying ATP
concentrations and constant
syntide-2 .
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conclusion:
inhibition was uncompetitive
with variable ATP
concentrations (parallel).
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Fig 5 Kinetics of CaMKII Inhibition by
NR2B.
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Conclusion:
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Fig C: inhibition was
competitive with
variable AC2
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Fig D: inhibition was
uncompetitive with
variable ATP
concentrations
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substrate :autocamtide2 (AC2)
Fig6 Nucleotides stabilize Ca2+-dependent
binding of CaMKII to NR2B.
with or without 100 μM ADP,
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Conclusion:
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Nonphosphorylated
CaMKII binds GST-NR2B
in a Ca2+concentrationdependent manner in the
presence of ADP.
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Not bind NR2B in the
absence of ADP (right).
DISCUSSION
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CaMKII Interaction with NR2B
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CaMKII Interaction with Densin
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CaMKII Interaction with α-actinin
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Implications for CaMKII signaling complexes
CaMKII Interaction with NR2B
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Autophosphorylation at Thr286 is also
necessary for interaction with a high
affinity binding site in NR2B corresponding
CaMKII Interaction with Densin
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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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