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Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons
Published on: April 6, 2011
Serotonin-induced cleavage of the atypical protein kinase C Apl III in Aplysia
Joanna K Bougie1, Diancai Cai, Margaret Hastings
1Department of Psychology, McGill University, Montreal Neurological Institute, Montreal, Quebec H3A 1B1, Canada.
Abstract:
A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.
Insights
Protein kinase Mζ (PKMζ) is crucial for long-lasting memory. In Aplysia, protein kinase Cζ (PKCζ) is cleaved to form PKMζ, a process essential for memory formation and maintenance.
Area of Science:
- Neuroscience
- Molecular Biology
- Cellular Neuroscience
Background:
- Protein kinase Mζ (PKMζ) is a proposed molecular substrate for long-term memory.
- PKMζ is synthesized differently in rodents and Aplysia, despite similar memory erasure by PKMζ inhibitors.
- In Aplysia, protein kinase Cζ (PKCζ) ortholog, PKC Apl III, is cleaved by calpain to form PKM.
Purpose of the Study:
- To investigate the mechanism of PKM Apl III formation in Aplysia.
- To determine if kinase activity is required for PKC Apl III cleavage.
- To examine the role of PKM Apl III in the late phase of intermediate-term facilitation.
Main Methods:
- Utilized a Förster resonance energy transfer (FRET) reporter to visualize PKC Apl III cleavage in live neurons.
- Applied serotonin stimulus to induce plasticity in sensory-motor neuron cocultures.
- Expressed dominant-negative PKM Apl III in motor neurons to assess its role in memory facilitation.
Main Results:
- Kinase activity is necessary for the calpain-mediated cleavage of PKC Apl III.
- Serotonin application induced PKC Apl III cleavage in motor neuron processes, dependent on calpain and protein synthesis.
- Cleavage was not observed in sensory neuron processes.
- Inhibition of PKM Apl III in motor neurons blocked intermediate-term facilitation.
Conclusions:
- PKC Apl III is cleaved into PKM Apl III during memory formation in Aplysia.
- The requirements for PKC Apl III cleavage mirror those for plasticity induction.
- PKM Apl III in motor neurons is essential for intermediate-term memory facilitation.
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