Effect of Zinc on Calmodulin‐Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

Abstract: The effect of increasing concentrations of Zn2+ (1 μM‐5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2‐M) fractions from rat cerebral cortex and purified calmodulinstimulated protein kinase II (CMKII). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2‐M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin‐stimulated conditions showed a dose‐dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 μM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin‐stimulated phosphoproteins in P2‐M and S3 showed biphasic behavior inhibition at <100 μM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the a and β subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin‐stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn2+‐stimulated, Ca2+/ calmodulin‐independent activity at concentrations of > 100 μM that produces a redistribution of activity biased toward autophosphorylation and an subunit with an altered mobility on sodium dodecyl sulfate‐containing gels.