Inhibition of Protein Phosphatase 1 Stimulates Secretion of Alzheimer Amyloid Precursor Protein

BackgroundAberrant metabolism of the Alzheimer amyloid precursor protein (APP) or its amyloidogenic Aβ fragment is thought to be centrally involved in Alzheimer’s disease. Nonamyloidogenic processing of APP involves its cleavage within the Aβ domain by a protease, termed α-secretase, and release of the large extracellular domain, termed APPS. Secretion of APPS can be stimulated by phorbol esters, activators of protein kinase C, with concurrent inhibition of Aβ production. While the role of protein kinases on APP metabolism has been investigated, considerably less effort has been devoted to elucidating the role played by protein phosphatases. Okadaic acid, a protein phosphatase inhibitor, has been shown to stimulate secretion of APPS, but the identity of the phosphatase involved has not been investigated.Materials and MethodsThe secretion of APPS from COS-1 cells was measured in the absence or presence of various doses of serme/threonine-specific phosphatase inhibitors. Quantitation of the derived IC50 values was used to determine the identity of the phosphatase involved in the control of APP secretion.ResultsThe availability of protein phosphatase inhibitors with different relative potencies against the different types of serine/threonine-specific protein phosphatase allowed us to examine which of the four known types of protein phosphatase might be involved in the regulation of APP secretion. Both okadaic acid and calyculin A stimulated the secretion of APP from COS-1 cells in a dose-dependent manner. The half-maximal dose for stimulation of APP secretion was approximately 100-fold higher with okadaic acid than with calyculin A.ConclusionsThe nearly 100-fold difference in the observed IC50 values for okadaic acid and calyculin A implicates a type 1 protein phosphatase in the control of APPS production. Protein phosphatase 1 (PP1) is known to be highly expressed in adult mammalian brain, both in neurons and glia. The identification of a specific phosphatase type in the control of APP secretion opens new avenues to the development of rational therapeutic intervention strategies aimed at the prevention and/or treatment of Alzheimer’s Disease.

[1]  D. Hartshorne,et al.  Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. , 1989, Biochemical and biophysical research communications.

[2]  P. Davies,et al.  SELECTIVE LOSS OF CENTRAL CHOLINERGIC NEURONS IN ALZHEIMER'S DISEASE , 1976, The Lancet.

[3]  G. Glenner,et al.  Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. , 1984, Biochemical and biophysical research communications.

[4]  Andreas Weidemann,et al.  Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein , 1989, Cell.

[5]  P. Greengard,et al.  Differential expression of protein phosphatase 1 isoforms in mammalian brain , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[6]  P. Greengard,et al.  Phosphorylation of DARPP-32 and protein phosphatase inhibitor-1 in rat choroid plexus: regulation by factors other than dopamine , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[7]  T. Sugimura,et al.  Identification of Members of the Protein Phosphatase 1 Gene Family in the Rat and Enhanced Expression of Protein Phosphatase 1α Gene in Rat Hepatocellular Carcinomas , 1990, Japanese journal of cancer research : Gann.

[8]  E. Masliah,et al.  Protein kinase C alteration is an early biochemical marker in Alzheimer's disease , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[9]  A. Nairn,et al.  Protein phosphatases: recent progress. , 1991, Advances in second messenger and phosphoprotein research.

[10]  M. Jennings,et al.  K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1. , 1993, The American journal of physiology.

[11]  E. F. da Cruz e Silva,et al.  Identification of a novel protein phosphatase catalytic subunit by cDNA cloning , 1988, FEBS letters.

[12]  P. Greengard,et al.  Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Greengard,et al.  Regulated cleavage of alzheimer β-amyloid precursor protein in the absence of the cytoplasmic tail , 1993, Neuroscience.

[14]  R. Katzman.,et al.  Reduced protein kinase C immunoreactivity and altered protein phosphorylation in Alzheimer's disease fibroblasts. , 1989, Archives of neurology.

[15]  J. Growdon,et al.  Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. , 1992, Science.

[16]  S. Younkin,et al.  Secretory processing of the Alzheimer amyloid beta/A4 protein precursor is increased by protein phosphorylation. , 1992, Biochemical and biophysical research communications.

[17]  P. Greengard,et al.  DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1 , 1984, Nature.

[18]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[19]  P. Greengard,et al.  Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/calmodulin-dependent protein kinase II. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Selkoe,et al.  Selective ectodomain phosphorylation and regulated cleavage of beta‐amyloid precursor protein. , 1994, The EMBO journal.

[21]  J. Casida,et al.  Protein phosphatase 2A and its [3H]cantharidin/[3H]endothall thioanhydride binding site. Inhibitor specificity of cantharidin and ATP analogues. , 1993, Biochemical pharmacology.

[22]  K. Dobkins,et al.  Decreased levels of protein kinase C in Alzheimer brain , 1988, Brain Research.

[23]  P. Greengard,et al.  Protein phosphorylation regulates secretion of Alzheimer beta/A4 amyloid precursor protein. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Tanzi,et al.  Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Takesada Mori,et al.  The possible involvement of protein phosphatase 1 in thrombin‐induced Ca2+ influx of human platelets , 1993, Journal of cellular biochemistry.

[26]  P. Greengard,et al.  Phosphorylation of alzheimer amyloid precursor protein by protein kinase C , 1992, Neuroscience.

[27]  P. Greengard,et al.  Activation of NMDA receptors induces dephosphorylation of DARPP-32 in rat striatal slices , 1990, Nature.

[28]  P. Greengard,et al.  The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  B. Hyman,et al.  Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid β protein precursor , 1993, Nature Genetics.

[30]  A. Takai,et al.  Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. , 1988, The Biochemical journal.

[31]  Paul Greengard,et al.  A dopamine- and cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions , 1983, Nature.

[32]  P. Greengard,et al.  Processing of Alzheimer beta/A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[33]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[34]  P. Greengard,et al.  Phosphorylation of DARPP‐32 Is Regulated by GABA in Rat Striatum and Substantia Nigra , 1994, Journal of neurochemistry.

[35]  B. Hyman,et al.  Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid beta protein precursor. , 1993, Nature genetics.