Calcium-promoted translocation of protein kinase C to synaptic membranes: Relation to the phosphorylation of an endogenous substrate (profein F1) involved in synaptic plasticity

The translocation of protein kinase C between membrane and cytosol has been implicated in several cellular processes (Kraft and Anderson, 1983; Wooten and Wrenn, 1984; Akers et al., 1985, 1986; Hirota et al., 1985; Wolf et al., 1986). We desired to identify potential trigger mechanisms underlying the translocation of protein kinase C activity to neural membranes following the synaptic plasticity observed after long-term potentiation (LTP; Akers et al., 1986). Takai et al. (1979) have suggested an important role for calcium in protein kinase C translocation; we have therefore studied the effects of Ca²⁺ on both the translocation of protein kinase C activity and the in vitro phosphorylation of its endogenous substrate, protein F1, in rat hippocampal synaptosomes. Since identical free Ca²⁺ levels were maintained in subsequent assays of synaptosomal membranes (SPM) and cytosol preparations, alterations in endogenous enzyme activity and in vitro phosphorylation were due to the Ca²⁺ present during treatment of synaptosomes, and not to the Ca²⁺ present during assays of enzymatic activity. This afforded the opportunity to relate directly such enzyme translocation to endogenous substrate phosphorylation. The major findings were as follows: 1. Following treatment of synaptosomes with Ca²⁺ protein kinase C activity in synaptic membrane and protein F1 in vitro phosphorylation were elevated in a dose-dependent manner. 2. The greatest increment in membrane protein kinase C activity and protein F1 in vitro phosphorylation occurred when Ca²⁺ was increased from 0.1 to 1.0 μM. Maximal levels of enzyme activity were seen following treatment with 10 μM Ca²⁺, and minimum levels were observed following treatment with EGTA. 3. Protein F1 phosphorylation was positively correlated with the protein kinase C activity found in membranes (r = 0.869;P < 0.001). 4. Treatment with Ca²⁺ increased the amount of protein kinase C activity in membranes and decreased the amount of kinase C activity in the soluble fraction. The sum total of synaptic membrane and soluble protein kinase C activities was similar in different preparations lysed with different Ca²⁺ levels, suggesting that a redistribution, or translocation, of protein kinase C had occurred. These results indicate that Ca²⁺ regulates the amount of protein kinase C activity associated with synaptic membranes. This association is directly correlated with protein F1 in vitro phosphorylation. since after LTP, protein kinase C activity is translocated to membranes (Akers et al., 1986) and membrane-bound protein F1 in vitro phosphorylation is elevated (Routtenberg et al., 1985), the translocation of protein kinase C to membranes, triggered by Ca²⁺, may account in part for the observed changes in protein kinase C and protein F1 following LTP.