Interactions between αCaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

Kato, Ken; Iwamoto, Toschitake; Kida, Satoshi

doi:10.1186/1756-6606-6-37

Cited by 7 publications

(4 citation statements)

References 43 publications

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“…Thus, at least in a crude way, the position of the catalytic region, as measured by FRET, is related to the activity of the enzyme. Direct comparison of the FRET signal of Camui with its enzymatic activity is consistent with this conclusion [ 14 , 15 , 17 ].…”

Section: Introductionsupporting

confidence: 67%

“…Understanding the Camui signal, however, requires a precise mapping of the conformational signal reported by the probe onto biochemically defined activation states. Previous work demonstrated a relationships of the Camui FRET signals to the biochemical states of CaMKII under in vitro conditions [ 14 , 15 , 17 , 31 ]. Based on this data, initial studies on the spines of living cells showed that the Camui signal after LTP induction was very short-lived—less than a minute—such that the major (~80%) component decayed with tau1 of ~5–6 sec [ 14 , 18 ].…”

Section: Discussionmentioning

confidence: 99%

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Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

2015

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Because CaMKII is the critical Ca2+ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca2+ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.

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Section: Introductionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

2015

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“…CaM then binds to the CaM-binding domain of CaMKII leading to its conformational change by dissociation of the auto-inhibitory domain from the catalytic domain. Furthermore, prolonged activation of CaMKII by the interaction of CaM-KII with CaM results in the autophosphorylation of threonine-286 (T286) (Kato et al 2013). Phosphorylation at T286 is thought to stabilize the CaM-bound form of CaMKII and therefore, prevents the inactivation of CaMKII kinase activity even after the dissociation of CaM.…”

Section: Camkii Mechanismsmentioning

confidence: 99%

Exercise-induced metabolic fluctuations influence AMPK, p38-MAPK and CaMKII phosphorylation in human skeletal muscle

et al. 2015

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During transition from rest to exercise, metabolic reaction rates increase substantially to sustain intracellular ATP use. These metabolic demands activate several kinases that initiate signal transduction pathways which modulate transcriptional regulation of mitochondrial biogenesis. The purpose of this study was to determine whether metabolic fluctuations per se affect the signaling cascades known to regulate peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). On two separate occasions, nine men performed a continuous (30-min) and an intermittent exercise (30 × 1-min intervals separated by 1-min of recovery) at 70% of . Skeletal muscle biopsies from the vastus lateralis were taken at rest and at +0 h and +3 h after each exercise. Metabolic fluctuations that correspond to exercise-induced variation in metabolic rates were determined by analysis of VO2 responses. During intermittent exercise metabolic fluctuations were 2.8-fold higher despite identical total work done to continuous exercise (317 ± 41 vs. 312 ± 56 kJ after intermittent and continuous exercise, respectively). Increased phosphorylation of AMP-activated protein kinase (AMPK) (˜2.9-fold, P < 0.01), calcium/calmodulin-dependent protein kinase II (CaMKII) (˜2.7-fold, P < 0.01) and p38-mitogen-activated protein kinase (MAPK) (˜4.2-fold, P < 0.01) occurred immediately in both exercises and to a greater extent after the intermittent exercise (condition x time interaction, P < 0.05). A single bout of intermittent exercise induces a greater activation of these signaling pathways regulating PGC-1α when compared to a single bout of continuous exercise of matched work and intensity. Chronic adaptations to exercise on mitochondria biogenesis are yet to be investigated.

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“…The Camui sensor is composed of CaMKIIα tagged with dVenus and GFP fluorophores on its N and C termini, respectively. Upon CaMKII activation, the distance between the termini increases, leading to a decrease in the Forster resonance energy transfer (FRET) between the fluorophores (see also [ 33 , 34 , 35 , 36 ]) and to an increase in GFP lifetime [ 32 ]. Therefore, change in the fluorescent lifetime of the Camui GFP reveals transition of CaMKII between its closed (inactive) and open (active) states [ 32 ].…”

Section: Introductionmentioning

confidence: 99%

Excitotoxic Insult Results in a Long-Lasting Activation of CaMKIIα and Mitochondrial Damage in Living Hippocampal Neurons

et al. 2015

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Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.

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Interactions between αCaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

Cited by 7 publications

References 43 publications

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

Exercise-induced metabolic fluctuations influence AMPK, p38-MAPK and CaMKII phosphorylation in human skeletal muscle

Excitotoxic Insult Results in a Long-Lasting Activation of CaMKIIα and Mitochondrial Damage in Living Hippocampal Neurons

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