PKC has been implicated in both short-term and long-term forms of synaptic plasticity which are believed to contribute to the physiological mechanisms of memory, and thus to behavioral and mental states. One widely held hypothesis for the persistence of changes in synaptic strength is that enzymes, such as PKC, which can enhance synaptic transmission in the short-term, become persistently active in the long-term. We have examined the regulation of PKC following a high-frequency tetanus of afferents in the CA1 region of the hippocampus that produces a long-term synaptic enhancement, LTP. We have observed that during LTP, PKC becomes persistently active through the proteolytic removal of the enzyme's autoinhibitory regulatory domain, increasing the level of the free, independently active catalytic fragment, termed PKM. This proteolytic activation, however, is specifically directed towards the ayptical zeta isoform of the enzyme, only one of the 10 isozymes of PKC. In addition, we have observed that low-frequency stimulation of afferents that produces LTD decreases the level of PKMzeta by proteolytic downregulation. The overall goal of this grant is to investigate how the unique properties of a typical PKCzeta, working in combination with other signal transduction pathways, regulate proteolytic processing to produce increases or decreases in PKMzeta during LTP and LTD, respectively. We will use a combination of biochemical and physiological techniques, both in vitro and in the hippocampal slice preparation, to address the following 3 Specific Aims: (1) to develop an assay to study the proteolytic processing of zeta. Preliminary results in vitro indicate that the calcium-dependent protease, calpain, can either form or degrade PKMzeta, depending upon the site of proteolytic cleavage on zeta. (2) To characterize the mechanisms that modulate this proteolytic processing. We have observed that the atypical isoform zeta, but not the conventional isoforms of PKC, has 2 potential CaM-kinase II phosphorylation sites in its catalytic domain. We will examine whether these, or other forms of post-translational modification of zeta, regulate the accessibility of zeta's cleavage sites to determine the direction of the change in PKMzeta. (3) To determine the functional consequences of the proteolytic activation of zeta on the long-term modulation of synaptic transmission. We have found that zeta phosphorylation can be isolated by the application of specific combinations of pharmacological agents. We will use these combinations in hippocampal slices to isolate the role of zeta phosphorylation during the maintenance of long-term synaptic plasticity.
These specific aims will help to achieve a detailed molecular and functional analysis of the proteolytic regulation of PKMzeta that causes both increases and decreases of constitutively active PKC during bidirectional synaptic plasticity.
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