Effect of zinc on calmodulin-stimulated protein kinase II and protein phosphorylation in rat cerebral cortex.
The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). 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 microM 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 less than 100 microM 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 alpha and beta 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 Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.
Weinberger
R P
RP
Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales, Australia.
Rostas
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000494:pharmacology*; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*; D019287:Zinc Sulfate
D002147:Calmodulin; D009419:Nerve Tissue Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D013431:Sulfates; D019287:Zinc Sulfate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015032:Zinc