a.j. robison et al., jbc papers published on september 19, 2005 ...
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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. - PowerPoint PPT PresentationTRANSCRIPT
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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
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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.
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CaMKII: Ca2+/calmodulin-dependent protein kinase II
•Autoregulatory :•Thr286-autophosphorylated CaMKII•Thr305/306-autophosphorylated CaMKII•Catalytic site : N-terminal
2002 Biochemical Society
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Thr286-autophosphorylated CaMKII
2002 Biochemical Society
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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:
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NATURE REVIEWS | NEUROSCIENCE OCTOBER 2004 www.nature.com/reviews/neuro
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J.M. Loftis, A. Janowsky / Pharmacology & Therapeutics 97 (2003) 55–85
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MATERIALS AND METHODS
Proteins Purified
GST Cosedimentation Assays
Kinase Assays
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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
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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
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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
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RESULTS
Figure1 A
Non-P [P-T286]
Ca2+/calmodulin-dependent
Sequential-P Basal-P: Basal Ca2+-
independent autophosphorylation [P-T286]
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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.
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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).
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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).
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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 .
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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 .
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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]
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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
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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
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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.
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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
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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)
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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,
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DISCUSSION
CaMKII Interaction with NR2B
CaMKII Interaction with Densin
CaMKII Interaction with α-actinin
Implications for CaMKII signaling complexes
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CaMKII Interaction with NR2B
Autophosphorylation at Thr286 is also necessary for interaction with a high affinity binding site in NR2B corresponding
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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.
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CaMKII Interaction with α-actinin Implications for CaMKII signaling
complexes.
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