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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.


Related in: MedlinePlus

Fear conditioning increases in vitro proteasome activity and phosphorylation of Rpt6-S120 in amygdala tissue. (A) Rats were exposed to several pairings of an auditory cue with a footshock or exposed to the shock (SK) or white noise (WN) individually and amygdala tissue collected 4 h later (n = 9–11 per group). Dotted line denotes 10 s in the training context, while solid lines denote 15 min in the training context. (B) Animals that received pairings of the white noise with the footshock froze significantly more during training than animals receiving the WN alone. (C–E) Proteasome chymotrypsin-like (C) trypsin-like (D) and peptidylglutamyl-peptide hydrolyzing-like (E) activities were increased only in rats that received the auditory cue paired with the footshock. (F) Fear conditioning increased phosphorylation of the proteasome regulatory subunit Rpt6 at Serine120 (G) There were no increases in the Rpt6 subunit but (H) fear conditioned animals showed enhanced protein polyubiquitination. *p < 0.05 from WN controls (B) or HC, SK and WN controls (C–H).
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Figure 2: Fear conditioning increases in vitro proteasome activity and phosphorylation of Rpt6-S120 in amygdala tissue. (A) Rats were exposed to several pairings of an auditory cue with a footshock or exposed to the shock (SK) or white noise (WN) individually and amygdala tissue collected 4 h later (n = 9–11 per group). Dotted line denotes 10 s in the training context, while solid lines denote 15 min in the training context. (B) Animals that received pairings of the white noise with the footshock froze significantly more during training than animals receiving the WN alone. (C–E) Proteasome chymotrypsin-like (C) trypsin-like (D) and peptidylglutamyl-peptide hydrolyzing-like (E) activities were increased only in rats that received the auditory cue paired with the footshock. (F) Fear conditioning increased phosphorylation of the proteasome regulatory subunit Rpt6 at Serine120 (G) There were no increases in the Rpt6 subunit but (H) fear conditioned animals showed enhanced protein polyubiquitination. *p < 0.05 from WN controls (B) or HC, SK and WN controls (C–H).

Mentions: Animals were trained to auditory fear conditioning as described previously (Jarome et al., 2011, 2012). Briefly, following 3 days of acclimation to the transporting and injection procedures, animals were placed in novel Context A and after a 6 min baseline, presented with 4 pairings of a white noise (72 dB, 10 s) with a footshock (1.0 mA, 1 s), 90-s ITI (intertrial interval). After a 4 min postshock period, the animals were removed from the chambers. Mircoinfusions were given immediately following the completion of the training session. For associative control experiments, animals were exposed to the white noise alone or underwent an immediate shock procedure (SK) as described previously (Jarome et al., 2011). Briefly, in the immediate shock procedure, animals were placed in Context A and immediately received 4 presentations of the footshock (1.0 mA, 1 s, 1-s ITI). Animals were then removed from the chamber following the final shock presentation. Animals are thus exposed to the shock but are unable to form a context-shock association using parameters such as these. In the white noise only control (WN), animals received an identical training session as the normal trained group except that the shock presentations were omitted. During training days for the associative control experiment (Figure 2), each of the four conditions (homecage, SK, WN, and trained) were equally represented during every batch of tissue collection. Order of conditioning for the associative control experiment was Trained, WN, SK, and then repeated.


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)

Fear conditioning increases in vitro proteasome activity and phosphorylation of Rpt6-S120 in amygdala tissue. (A) Rats were exposed to several pairings of an auditory cue with a footshock or exposed to the shock (SK) or white noise (WN) individually and amygdala tissue collected 4 h later (n = 9–11 per group). Dotted line denotes 10 s in the training context, while solid lines denote 15 min in the training context. (B) Animals that received pairings of the white noise with the footshock froze significantly more during training than animals receiving the WN alone. (C–E) Proteasome chymotrypsin-like (C) trypsin-like (D) and peptidylglutamyl-peptide hydrolyzing-like (E) activities were increased only in rats that received the auditory cue paired with the footshock. (F) Fear conditioning increased phosphorylation of the proteasome regulatory subunit Rpt6 at Serine120 (G) There were no increases in the Rpt6 subunit but (H) fear conditioned animals showed enhanced protein polyubiquitination. *p < 0.05 from WN controls (B) or HC, SK and WN controls (C–H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757295&req=5

Figure 2: Fear conditioning increases in vitro proteasome activity and phosphorylation of Rpt6-S120 in amygdala tissue. (A) Rats were exposed to several pairings of an auditory cue with a footshock or exposed to the shock (SK) or white noise (WN) individually and amygdala tissue collected 4 h later (n = 9–11 per group). Dotted line denotes 10 s in the training context, while solid lines denote 15 min in the training context. (B) Animals that received pairings of the white noise with the footshock froze significantly more during training than animals receiving the WN alone. (C–E) Proteasome chymotrypsin-like (C) trypsin-like (D) and peptidylglutamyl-peptide hydrolyzing-like (E) activities were increased only in rats that received the auditory cue paired with the footshock. (F) Fear conditioning increased phosphorylation of the proteasome regulatory subunit Rpt6 at Serine120 (G) There were no increases in the Rpt6 subunit but (H) fear conditioned animals showed enhanced protein polyubiquitination. *p < 0.05 from WN controls (B) or HC, SK and WN controls (C–H).
Mentions: Animals were trained to auditory fear conditioning as described previously (Jarome et al., 2011, 2012). Briefly, following 3 days of acclimation to the transporting and injection procedures, animals were placed in novel Context A and after a 6 min baseline, presented with 4 pairings of a white noise (72 dB, 10 s) with a footshock (1.0 mA, 1 s), 90-s ITI (intertrial interval). After a 4 min postshock period, the animals were removed from the chambers. Mircoinfusions were given immediately following the completion of the training session. For associative control experiments, animals were exposed to the white noise alone or underwent an immediate shock procedure (SK) as described previously (Jarome et al., 2011). Briefly, in the immediate shock procedure, animals were placed in Context A and immediately received 4 presentations of the footshock (1.0 mA, 1 s, 1-s ITI). Animals were then removed from the chamber following the final shock presentation. Animals are thus exposed to the shock but are unable to form a context-shock association using parameters such as these. In the white noise only control (WN), animals received an identical training session as the normal trained group except that the shock presentations were omitted. During training days for the associative control experiment (Figure 2), each of the four conditions (homecage, SK, WN, and trained) were equally represented during every batch of tissue collection. Order of conditioning for the associative control experiment was Trained, WN, SK, and then repeated.

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.


Related in: MedlinePlus