IDENTIFICATION OF A MAJOR AUTOPHOSPHORYLATION SITE ON POSTSYNAPTIC DENSITY-ASSOCIATED CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE/

One of the most abundant proteins in postsynaptic densities is identic al or very similar to the ar subunit of the Ca2+/calmodulin-dependent protein kinase II. Autophosphorylation of this protein in isolated pos tsynaptic densities was studied under various conditions, following in hibition of endogenous phosphatase activity with microcystin-LR. Phosp horylation accompanied by a shift in the enzyme's electrophoretic mobi lity was observed upon incubation with Ca2+ and calmodulin at 37 degre es C. Brief incubation with Ca2+ and calmodulin at 0 degrees C resulte d in a low level of phosphorylation and no change in mobility, Followi ng this limited Ca2+-dependent phosphorylation, however, a high level of phosphorylation could be achieved in the absence of Ca2+, upon incu bation at 37 degrees C. Comparison of reverse-phase HPLC phosphopeptid e elution profiles obtained following phosphorylation at 37 degrees C, in the presence and absence of Ca2+, as described above, showed diffe rences, suggesting that certain distinct sites may be phosphorylated u nder each condition, A major phosphopeptide peak, however, with the am ino acid sequence Met-Leu-Thr(P)-Ile Asn-Pro Ser-Lys was identified un der both conditions, This sequence is identical to the predicted seque nce containing Thr-253 of the Ca2+/calmodulin-dependent protein kinase II, The results suggest that phosphorylation at Thr-253 requires an i nitial Ca2+-dependent phosphorylation, which may be at a different sit e, but does not depend on the continued presence of Ca2+ to proceed. T he observed mode of regulation of autophosphorylation at Thr-253 appea rs to be unique to the postsynaptic density-associated enzyme.