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CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories.

Jarome TJ, Kwapis JL, Ruenzel WL, Helmstetter FJ - Front Behav Neurosci (2013)

Bottom Line: We found increases in the phosphorylation of proteasome ATPase subunit Rpt6 at Serine-120 and an enhancement in proteasome activity in the amygdala following fear conditioning.Pharmacological manipulation of CaMKII, but not PKA, in vivo significantly reduced both the learning-induced increase in Rpt6 Serine-120 phosphorylation and the increase in proteasome activity without directly affecting protein polyubiquitination levels.These results indicate a novel role for CaMKII in memory formation through its regulation of protein degradation and suggest that CaMKII regulates Rpt6 phosphorylation and proteasome function both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Wisconsin-Milwaukee Milwaukee, WI, USA.

ABSTRACT
CaMKII and Protein Kinase A (PKA) are thought to be critical for synaptic plasticity and memory formation through their regulation of protein synthesis. Consistent with this, numerous studies have reported that CaMKII, PKA and protein synthesis are critical for long-term memory formation. Recently, we found that protein degradation through the ubiquitin-proteasome system is also critical for long-term memory formation in the amygdala. However, the mechanism by which ubiquitin-proteasome activity is regulated during memory formation and how protein degradation interacts with known intracellular signaling pathways important for learning remain unknown. Recently, evidence has emerged suggesting that both CaMKII and PKA are capable of regulating proteasome activity in vitro through the phosphorylation of proteasome regulatory subunit Rpt6 at Serine-120, though whether they regulate Rpt6 phosphorylation and proteasome function in vivo remains unknown. In the present study we demonstrate for the first time that fear conditioning transiently modifies a proteasome regulatory subunit and proteasome catalytic activity in the mammalian brain in a CaMKII-dependent manner. We found increases in the phosphorylation of proteasome ATPase subunit Rpt6 at Serine-120 and an enhancement in proteasome activity in the amygdala following fear conditioning. Pharmacological manipulation of CaMKII, but not PKA, in vivo significantly reduced both the learning-induced increase in Rpt6 Serine-120 phosphorylation and the increase in proteasome activity without directly affecting protein polyubiquitination levels. These results indicate a novel role for CaMKII in memory formation through its regulation of protein degradation and suggest that CaMKII regulates Rpt6 phosphorylation and proteasome function both in vitro and in vivo.

No MeSH data available.


CaMKII, but not PKA, regulates increases in proteasome activity following fear conditioning. Rats received infusions of a PKA inhibitor (myr-PKI), CaMKII inhibitor (myr-AIP) or vehicle immediately after fear conditioning and amygdala tissue was collected 4 h later (n = 8–10 per group). (A) Inhibiting PKA had no effect on proteasome activity, while inhibiting CaMKII significantly reduced proteasome chymotrypsin-like activity relative to vehicle infused trained rats. (B) Inhibiting CaMKII, but not PKA, reduced proteasome peptidylglutamyl-peptide hydrolyzing-like activity. (C) Neither inhibitor altered proteasome trypsin-like activity. *p < 0.05 from vehicle. #P = 0.07 from vehicle.
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Figure 4: CaMKII, but not PKA, regulates increases in proteasome activity following fear conditioning. Rats received infusions of a PKA inhibitor (myr-PKI), CaMKII inhibitor (myr-AIP) or vehicle immediately after fear conditioning and amygdala tissue was collected 4 h later (n = 8–10 per group). (A) Inhibiting PKA had no effect on proteasome activity, while inhibiting CaMKII significantly reduced proteasome chymotrypsin-like activity relative to vehicle infused trained rats. (B) Inhibiting CaMKII, but not PKA, reduced proteasome peptidylglutamyl-peptide hydrolyzing-like activity. (C) Neither inhibitor altered proteasome trypsin-like activity. *p < 0.05 from vehicle. #P = 0.07 from vehicle.

Mentions: Proteasome activity assays were performed as described previously with a small scale modification (Lopez-Salon et al., 2001; Ehlers, 2003; Upadhya et al., 2006). Samples (50μg, Figure 4; 100μg, Figures 1, 2) were diluted in DDH2O and mixed with reaction buffer (250 mM HEPES, pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.01% SDS, 5 mM ATP). Fluorogenic peptides Suc-LLVY-AMC (Millipore, Billerica, MA), Bz-VGR-AMC or z-LLE-AMC (Enzo Life Sciences, Farmingdale, NY) were added to the samples to assess proteasome chymotrypsin-like, trypsin-like and peptidylglutamyl-like activities, respectively (10 μM). The reaction was incubated at 37°C for 30 min (Bz-VGR-AMC and z-LLE-AMC) or 2 h (Suc-LLVY-AMC) and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT). Protein free blanks were used and an AMC standard curve was produced according to the manufacturer's instructions.


CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories.

Jarome TJ, Kwapis JL, Ruenzel WL, Helmstetter FJ - Front Behav Neurosci (2013)

CaMKII, but not PKA, regulates increases in proteasome activity following fear conditioning. Rats received infusions of a PKA inhibitor (myr-PKI), CaMKII inhibitor (myr-AIP) or vehicle immediately after fear conditioning and amygdala tissue was collected 4 h later (n = 8–10 per group). (A) Inhibiting PKA had no effect on proteasome activity, while inhibiting CaMKII significantly reduced proteasome chymotrypsin-like activity relative to vehicle infused trained rats. (B) Inhibiting CaMKII, but not PKA, reduced proteasome peptidylglutamyl-peptide hydrolyzing-like activity. (C) Neither inhibitor altered proteasome trypsin-like activity. *p < 0.05 from vehicle. #P = 0.07 from vehicle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757295&req=5

Figure 4: CaMKII, but not PKA, regulates increases in proteasome activity following fear conditioning. Rats received infusions of a PKA inhibitor (myr-PKI), CaMKII inhibitor (myr-AIP) or vehicle immediately after fear conditioning and amygdala tissue was collected 4 h later (n = 8–10 per group). (A) Inhibiting PKA had no effect on proteasome activity, while inhibiting CaMKII significantly reduced proteasome chymotrypsin-like activity relative to vehicle infused trained rats. (B) Inhibiting CaMKII, but not PKA, reduced proteasome peptidylglutamyl-peptide hydrolyzing-like activity. (C) Neither inhibitor altered proteasome trypsin-like activity. *p < 0.05 from vehicle. #P = 0.07 from vehicle.
Mentions: Proteasome activity assays were performed as described previously with a small scale modification (Lopez-Salon et al., 2001; Ehlers, 2003; Upadhya et al., 2006). Samples (50μg, Figure 4; 100μg, Figures 1, 2) were diluted in DDH2O and mixed with reaction buffer (250 mM HEPES, pH 7.5, 5 mM EDTA, 0.5% NP-40, 0.01% SDS, 5 mM ATP). Fluorogenic peptides Suc-LLVY-AMC (Millipore, Billerica, MA), Bz-VGR-AMC or z-LLE-AMC (Enzo Life Sciences, Farmingdale, NY) were added to the samples to assess proteasome chymotrypsin-like, trypsin-like and peptidylglutamyl-like activities, respectively (10 μM). The reaction was incubated at 37°C for 30 min (Bz-VGR-AMC and z-LLE-AMC) or 2 h (Suc-LLVY-AMC) and fluorescence monitored at 360 (excitation)/460 (emission) on a monochromatic plate reader (Synergy H1; Biotek, Winooski, VT). Protein free blanks were used and an AMC standard curve was produced according to the manufacturer's instructions.

Bottom Line: We found increases in the phosphorylation of proteasome ATPase subunit Rpt6 at Serine-120 and an enhancement in proteasome activity in the amygdala following fear conditioning.Pharmacological manipulation of CaMKII, but not PKA, in vivo significantly reduced both the learning-induced increase in Rpt6 Serine-120 phosphorylation and the increase in proteasome activity without directly affecting protein polyubiquitination levels.These results indicate a novel role for CaMKII in memory formation through its regulation of protein degradation and suggest that CaMKII regulates Rpt6 phosphorylation and proteasome function both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Wisconsin-Milwaukee Milwaukee, WI, USA.

ABSTRACT
CaMKII and Protein Kinase A (PKA) are thought to be critical for synaptic plasticity and memory formation through their regulation of protein synthesis. Consistent with this, numerous studies have reported that CaMKII, PKA and protein synthesis are critical for long-term memory formation. Recently, we found that protein degradation through the ubiquitin-proteasome system is also critical for long-term memory formation in the amygdala. However, the mechanism by which ubiquitin-proteasome activity is regulated during memory formation and how protein degradation interacts with known intracellular signaling pathways important for learning remain unknown. Recently, evidence has emerged suggesting that both CaMKII and PKA are capable of regulating proteasome activity in vitro through the phosphorylation of proteasome regulatory subunit Rpt6 at Serine-120, though whether they regulate Rpt6 phosphorylation and proteasome function in vivo remains unknown. In the present study we demonstrate for the first time that fear conditioning transiently modifies a proteasome regulatory subunit and proteasome catalytic activity in the mammalian brain in a CaMKII-dependent manner. We found increases in the phosphorylation of proteasome ATPase subunit Rpt6 at Serine-120 and an enhancement in proteasome activity in the amygdala following fear conditioning. Pharmacological manipulation of CaMKII, but not PKA, in vivo significantly reduced both the learning-induced increase in Rpt6 Serine-120 phosphorylation and the increase in proteasome activity without directly affecting protein polyubiquitination levels. These results indicate a novel role for CaMKII in memory formation through its regulation of protein degradation and suggest that CaMKII regulates Rpt6 phosphorylation and proteasome function both in vitro and in vivo.

No MeSH data available.