Calcium-Stimulated Protein Phosphorylation in Synaptic Membranes

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Abstract
Synaptic membranes from rat brain contain several Ca-requiring protein kinase (PK) activities with different substrate specificities: an activity (CaH-PK) effective at high concentrations of Ca2+ in the absence of Mg2+ (active on class F substrates); a (Ca + Mg)-PK activity that is mediated by Ca2+ in the presence of Mg2+ (active on class B substrates); (Ca-CaM)-PK activities that exhibit simultaneous requirements for both Ca2+ and CaM calmodulin (for class C and D substrates). Also described are 3 activities that do not require Ca2+; a Mg-PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ alone (Mg-PK activity) and is unaffected by the addition of Ca2+ and CaM, the substrates of which show different responses to several types of inhibitors; and the previously well characterized cAMP-dependent PK activities. Several of the substrates of these kinases are identified. Among them are P43 (class A), as the .alpha. subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75-P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca2+ and CaM. The half-maximal stimulation (K0.5) for Ca2+ in the presence of CaM was 1.0 .mu.M Ca2+F in untreated membranes. There is little change in this value on prior EGTA [ethylene glycol bis(.beta.-aminoethylether) tetraacetate] extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by .gtoreq. 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ was 0.4 .mu.g per reaction mixture (8 .mu.g/ml) or 1.35 .mu.g per reaction mixture (27 .mu.g/ml), for the untreated and EGTA-treated membranes, respectively.