No abstract

red fluorescent protein; PB1, Phox and Bem 1; PBS, phosphate-buffered saline; PDK, phosphoinositide-dependent kinase; PI3K, phosphoinositide-3 kinase; PKC, protein kinase C; PKM, protein kinase M. AbstractIn vertebrates, a brain-specific transcript from the atypical protein kinase C (PKC) f gene encodes protein kinase M (PKM) f, a constitutively active kinase implicated in the maintenance of synaptic plasticity and memory. We have cloned the atypical PKC from Aplysia, PKC Apl III. We did not find a transcript in Aplysia encoding PKMf, and evolutionary analysis of atypical PKCs suggests formation of this transcript is restricted to vertebrates. Instead, over-expression of PKC Apl III in Aplysia sensory neurons leads to production of a PKM fragment of PKC Apl III. This cleavage was induced by calcium and blocked by calpain inhibitors. Moreover, nervous system enriched spliced forms of PKC Apl III show enhanced cleavage. PKC Apl III could also be activated through phosphorylation downstream of phosphoinositide 3-kinase. We suggest that PKM forms of atypical PKCs play a conserved role in memory formation, but the mechanism of formation of these kinases has changed over evolution.

SUMMARY Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant negative atypical PKM selectively reversed associative LTF, while a dominant negative classical PKM selectively reversed non-associative LTF. While both PKMs are formed from calpain-mediated cleavage of PKCs, each form of LTF is sensitive to a distinct dominant negative calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dominant negative classical calpain, while non-associative LTF is blocked by dominant negative small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories.

A constitutively active kinase, known as protein kinase M (PKM), is proposed to act as a long-lasting molecular memory trace. While PKM is formed in rodents through translation of a transcript initiating in an intron of the protein kinase C (PKC) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKM erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKC, 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.