Ca(2+)-induced persistent protein kinase C activation in rat hippocampal homogenates.
Protein kinase C (PKC) is thought to play an important role in neuronal function by mediating changes in synaptic strength. Specifically, it has been argued that persistent PKC activation underlies the maintenance of long-term potentiation (LTP) of synaptic transmission in the hippocampus, a model widely used to study mammalian learning and memory. Because the induction of LTP is known to be dependent upon Ca2+ influx into the postsynaptic neuron, we investigated Ca(2+)-dependent mechanisms that operate to elicit persistent PKC activation in the hippocampus. Hippocampal homogenates were incubated with Ca2+ for a brief period and subsequently assayed for persistent changes in basal (Ca(2+)-independent) PKC activity, using the selective PKC substrate neurogranin(28-43) (NG(28-43)). After Ca2+ incubation, basal PKC phosphorylation of NG(28-43) was increased and expression of the increased activity could be inhibited by PKC(19-36), a selective peptide inhibitor of PKC. These data indicate the presence of a persistently activated form of PKC in Ca(2+)-pretreated hippocampal homogenates. The persistently activated PKC was localized to the soluble fraction of homogenates. Generation of the soluble, persistently activated form of PKC was blocked by the calpain inhibitor, leupeptin, suggesting a proteolytic activation of PKC. Column chromatography and Western blots indicated the presence of PKM, a proteolytic fragment of PKC that is active in the absence of calcium, diacylglycerols, or phospholipid cofactors. Thus, Ca2+ induces proteolytic activation of PKC in hippocampal homogenates. This suggests that proteolytic activation is a plausible candidate as a mechanism underlying the persistent activation of PKC associated with LTP.
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030.
J S
JS
Sessoms
S J
SJ
Chen
D M
DM
Chetkovich
C M
CM
Powell
E D
ED
Roberson
J D
JD
Sweatt
E
E
Klann
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002148:Calmodulin-Binding Proteins; D002852:Chromatography, Ion Exchange; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D006624:Hippocampus / Q000187:drug effects / Q000201:enzymology*; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D009419:Nerve Tissue Proteins; D051579:Neurogranin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D002148:Calmodulin-Binding Proteins; D007976:Leupeptins; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D051579:Neurogranin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C032854:leupeptin; D002118:Calcium