Amyloid beta peptide formation in cell-free preparations. Regulation by protein kinase C, calmodulin, and calcineurin.
Amyloid beta peptide (Abeta) is a short peptide that is the major constituent of the amyloid plaques and cerebrovascular amyloid deposits found in Alzheimer's disease. The lack of availability of a cell-free system in which to study Abeta formation has limited our understanding of the molecular mechanisms involved in its production. We report here the reconstitution of such a cell-free system. The reconstituted Abeta formation was temperature-dependent and required ATP. Preincubation with purified protein kinase C (PKC) induced a pronounced inhibition of Abeta formation, similar to that observed in intact cells upon stimulation of PKC. The calmodulin antagonists W-7 and trifluoperazine inhibited Abeta formation and enhanced the action of PKC in both the cell-free system and intact cells. A role for the calcium/calmodulin-activated protein phosphatase calcineurin in the regulation of Abeta formation was demonstrated using a specific peptide inhibitor of calcineurin in vitro as well as cyclosporin A, a cell-permeant inhibitor of calcineurin, in intact cells. Our results suggest that a single substrate might mediate opposing actions of PKC and calcineurin in the regulation of Abeta formation.
Desdouits
F
F
Laboratory of Molecular and Cellular Neuroscience and the Zachary and Elizabeth M. Fisher Center for Research on Alzheimer Disease, The Rockefeller University, New York, New York 10021, USA.
Buxbaum
J D
JD
Desdouits-Magnen
J
J
Nairn
A C
AC
Greengard
P
P
D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D019703:Calcineurin; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002474:Cell-Free System; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D011493:Protein Kinase C; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases