Calcium-stimulated protein phosphorylation in synaptic membranes

Synaptic membranes from rat brain contain several calcium-requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (CaH-PK) effective at high concentrations of Ca2+ ion in the absence of Mg2+ (active on class F substrates); (b) a (Ca + Mg)-PK activity that is mediated by Ca2+ ion in the presence of Mg2+ (active on class B substrates); (c) (Ca-CaM)-PK activities that exhibit simultaneous requirements for both Ca2+ ion and CaM (for class C and D substrates). Also described are three activities (d-f) that do not require Ca2+ ion: (d) a Mg-PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ ion alone (Mg-PK activity) and is unaffected by the addition of Ca2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP-dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: 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 found to be 1.0 microM Ca2+F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by greater than or equal to 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ ion was found to equal 0.4 microgram per reaction mixture (8 micrograms/ml) or 1.35 micrograms per reaction mixture (27 micrograms/ml), for the untreated and EGTA-treated membranes, respectively.