Inhibition of endogenous phosphatase in a postsynaptic density fraction allows extensive phosphorylation of the major postsynaptic density protein.
The major postsynaptic density protein, proposed to be a calcium/calmodulin-dependent protein kinase, becomes phosphorylated when a postsynaptic density preparation from rat cerebral cortex is incubated in medium containing calcium and calmodulin. Upon longer incubation, however, the level of phosphorylation declines, suggesting the presence of a phosphatase activity. When Microcystin-LR, a phosphatase inhibitor, is included in the phosphorylation medium, the decline in phosphorylation is prevented and a higher maximal level of phosphorylation can be achieved. Under these conditions, the maximal phosphorylation of major postsynaptic density protein is accompanied by a nearly complete shift in its electrophoretic mobility from 50 kDa to 54 kDa, similar to that described for the alpha subunit of the soluble calcium/calmodulin-dependent protein kinase II. Of the four major groups of serine/threonine protein phosphatases, the enzyme responsible for the dephosphorylation of major postsynaptic density protein is neither type 2C, which is insensitive to Microcystin-LR, nor type 2B, which is calcium-dependent. As Microcystin-LR is much more potent than okadaic acid in inhibiting the dephosphorylation of major postsynaptic density protein, it is likely that the postsynaptic density-associated phosphatase is a type 1. The above results indicate that the relatively low level of phosphorylation of the major postsynaptic density protein observed in preparations containing postsynaptic densities is not due to a difference between the cytoplasmic and postsynaptic density-associated calcium/calmodulin-dependent kinases as previously proposed, but to a phosphatase activity, presumably belonging to the type 1 group.
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
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Dosemeci
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Reese
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000378:metabolism* / Q000648:ultrastructure; D004533:Egtazic Acid / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D008387:Marine Toxins; D052998:Microcystins; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019319:Okadaic Acid; D010456:Peptides, Cyclic / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism* / Q000648:ultrastructure
D002147:Calmodulin; D004988:Ethers, Cyclic; D008387:Marine Toxins; D052998:Microcystins; D009419:Nerve Tissue Proteins; D010456:Peptides, Cyclic; C033652:postsynaptic density proteins; D019319:Okadaic Acid; D004533:Egtazic Acid; D010744:Phosphoric Monoester Hydrolases; C057862:cyanoginosin LR; D002118:Calcium