A. Dosemeci et al., IDENTIFICATION OF A MAJOR AUTOPHOSPHORYLATION SITE ON POSTSYNAPTIC DENSITY-ASSOCIATED CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE/, The Journal of biological chemistry, 269(50), 1994, pp. 31330-31333
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.