A comparative genomic analysis of atypical PKC performed by Wayne Sossin and colleagues at McGill University found that the translational mechanism for the formation of PKMζ, the hallmark of which is a conserved methionine in the hinge region that initiates the synthesis of PKMζ [65], arose around the time of the gene duplication of the single invertebrate aPKC gene into the two vertebrate aPKC isoforms, ζ and ι/λ [93]. These two isoforms, whose actions can be similar in neurons [47], are the two most closely related genes of the 9-member PKC gene family. Extending this analysis, Ling Pan (SUNY Downstate) and I found that the lamprey, an early, jawless cyclostome vertebrate, has an apparent single aPKC, with features of both PKCζ and PKCι/λ, that contains the hallmark hinge methionine found in PKMζ that initiates translation of the independent catalytic domain. Therefore, the formation of atypical PKM by new protein synthesis originated at or before the splitting of cyclostomes from the main vertebrate line of evolution (the cyclostome–gnathostome split). This establishes the origin of the formation of PKM by new protein synthesis, and therefore the mechanism maintaining late-LTP, at least ~500 million years ago in the Cambrian period [94, 95].

Remarkably, a persistently active PKM form is also generated from the invertebrate aPKC, which lacks the vertebrate PKMζ translational start site [93], and this atypical PKM plays fundamental roles in long-term memory maintenance in widely divergent invertebrate phyla. Working with the arthropod Drosophila melanogaster, Jerry Yin and our colleagues at the University of Wisconsin at Madison showed that the persistent activity of atypical PKM is both necessary and sufficient for long-term memory of olfactory avoidance behavior that is induced by associative conditioning [96]. Drosophila atypical PKM is enriched in the fly head [96], just as PKMζ is specifically expressed in neural tissue [65, 97], but the mechanism for the formation of atypical PKM in Drosophila has not yet been elucidated. In the mollusk Aplysia californica, David Glanzman and colleagues at UCLA found that the persistent activity of atypical PKM is crucial for maintaining behavioral long-term sensitization of withdrawal reflexes as late as 7 days after training, well beyond the initial, protein synthesis-dependent consolidation phase for the sensitization [98]. In addition, Glanzman found that the Aplysia orthologue of PKMζ also maintains the long-term synaptic facilitation of sensorimotor synapses that mediates the behavior [98]. As shown by Sossin and colleagues, proteolysis of aPKC is critical for the formation of atypical PKM in Aplysia, and the proteolytic formation of atypical PKM by sensitizing stimulation requires both the protease calpain and new protein synthesis [93, 99]. How long-term memory maintained by atypical PKM in Aplysia might require both new protein synthesis and proteolysis is not yet known, but possibilities include new synthesis of the precursor aPKC, of the protease that cleaves the aPKC, or of another molecule that facilitates the cleavage or stabilizes the atypical PKM [99]. Eric Kandel and his colleagues at Columbia University have shown that the translation factor, Aplysia cytoplasmic polyadenylation element binding protein (CPEB) that has prion-like properties of self-perpetuation [100, 101] is required for sustaining long-term facilitation during a persistent, protein synthesis-dependent period lasting ~ 2 days [102]. Because Aplysia atypical PKM also maintains long-term facilitation during this period [98], CPEB may interact with atypical PKM, either by regulating the synthesis of aPKC or the protease that cleaves this precursor to PKM, or, conversely, as a mechanism regulated by PKM.

In both Drosophila and rats, overexpression of PKMζ enhances long-term memory. Jerry Yin and our colleagues demonstrated that transgenic flies overexpressing either mouse PKMζ or the Drosophila atypical PKM have stronger long-term memory, and therefore the mechanism for memory enhancement by increasing PKMζ activity, like that of memory erasure by decreasing PKMζ activity, is evolutionarily conserved [96]. Furthermore, by transfecting PKMζ into the neocortex of rats, Yadin Dudai and our colleagues at the Weizmann Institute showed that not only are new memories strengthened when PKMζ is overexpressed before training, but even old, faded memories are robustly enhanced when the kinase is overexpressed a week after training [25]. The mechanisms by which increasing PKMζ by overexpression enhances memory in both vertebrates and invertebrates are not known, but may involve upregulation of the positive feedback loops of local translation and “synaptic autotagging” that have been proposed to maintain the synaptic compartmentalization of PKMζ [59], as discussed in the next section.

Why is the persistently active PKM form of an atypical PKC crucial for memory maintenance, whether it is generated by cleavage of full-length PKC as in Aplysia, or by transcription from an internal promoter within the PKCζ gene as in vertebrates? Although one can only speculate, a clue may be the original function of aPKC in cells. Single cell organisms such as yeast express a single PKC, but multicellular animals express multiple PKC isoforms generated by gene duplication. In C. elegans, the function of aPKC has already specialized to establish and maintain apical compartments within polarized cells through participation in a highly conserved multiprotein complex, called the anterior PAR complex (for par titioning), consisting of the adapter proteins PAR6 and PAR3, the small GTPase Cdc42, and aPKC [103] (Figure 2A). In this apically localized complex, Cdc42 receives extracellular signals and stimulates PAR6, which then binds to the regulatory domain of aPKC, activating the kinase [104]. The PAR complex is conserved in polarized cells throughout evolution and defines the anterior pole of the C. elegans embryo, the apical domain of Drosophila neuroblasts to control their asymmetric division, and the apical membrane of epithelial cells to promote apical-basal polarity and the formation and maintenance of cell–cell junctions [103, 105–107]. Although the mechanisms by which the PAR complex mediate polarity are only beginning to be elucidated, a genome-wide screen in C. elegans has shown that the complex directs the trafficking of membrane proteins through the regulation of endocytosis and vesicle recycling [108, 109]. This mechanism is evolutionarily conserved because it is also observed in human HeLa cells [108].

Figure 2 Model of PKMζ-mediated LTP maintenance as a specialized form of aPKC regulation of cell polarity. A) In polarized cells such as epithelial cells, polarity signals activate PAR6, which binds to the aPKC regulatory domain (red) and activates the enzyme. Phosphorylation by aPKC then traffics membrane proteins to the apical compartment of the polarized cell. B) In spines, PKMζ is synthesized after LTP induction or learning and potentiates synaptic strength by NSF-dependent trafficking of AMPARs to the PSD, the apical compartment of the postsynaptic spine. The absence of a PKCζ regulatory domain isolates PKMζ from other postsynaptic signaling, allowing the kinase to store long-term information without interference from short-term synaptic events. PKMζ maintains both synaptic potentiation and its own localization at the synapse by forming positive feedback loops, involving binding of PKMζ to postsynaptic GluA2 subunit-containing AMPAR-binding proteins, such as PICK1 and KIBRA. The persistent activity of postsynaptic PKMζ is required to maintain decreased AMPAR endocytosis, preventing both AMPAR and kinase elimination from the potentiated synapse. Other positive feedback loops, such as that involving PIN1, maintain increases in the amount of PKMζ through enhanced local translation. Full size image

The general function of aPKC to distribute membrane proteins to apical compartments may have adapted to control the trafficking of glutamate receptors to the postsynaptic density, the apical compartment of the synaptic spine (Figure 2B). Atypical PKC may originally have participated in development of the synapse. Indeed, roles for PKMζ in synaptic maturation and dendritic development have recently been described [91, 110].

Once established as a mechanism for trafficking glutamate receptors to the synapse during development, the further activation of full-length aPKC might have been useful for short-term synaptic plasticity and short-term memory. Then, mutations that either allow proteolysis in the hinge between the regulatory and catalytic domains in invertebrates [99], or that generate independent translation of the catalytic domain in vertebrates [65], would have transformed this short-term memory mechanism into a long-term memory mechanism (Figure 2B).

The truncation of the aPKC regulatory domain to form an independent catalytic domain would serve two purposes in a molecular mechanism of long-term memory (Figure 2B). First, the enzymatic activity of aPKC becomes persistent, because of the removal of the autoinhibitory pseudosubstrate of the regulatory domain, as described above. Second, the regulation of this persistent atypical PKM activity becomes functionally isolated from the extracellular signaling that is normally transmitted into the cell by the other PAR proteins and second messengers that activate the full-length kinase by binding to the aPKC regulatory domain. Thus, once formed, the autonomous activity of atypical PKM that maintains long-term memory is independent from the transient signal transduction events that regulate short-term synaptic potentiation or depression. This feature may be important if long-term information about experiences in the past is to be stored in the same neural circuitry that is continually modified by short-term experiences in the present.