Calcium-promoted translocation of protein kinase C to synaptic membranes: relation to the phosphorylation of an endogenous substrate (protein 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 microM. Maximal levels of enzyme activity were seen following treatment with 10 microM Ca2+, and minimum levels were observed following treatment with EGTA.