Notizen: Abstract: We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phospha-tase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p-hydroxymercuri-benzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP-depen-dent protein kinase (cAMP-dPK) activity was present in both supernatant (S2) and paniculate (P2) fractions, with the majority (60–85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP-dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory sub-unit of the cAMP-dPK, R-II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP-dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were detected, suggesting that the genetic defect in cAMP metabolism is not accompanied by constituent abnormalities in phosphorylated substrates in the adult fly, and that the physiological defects in these mutants result from aberrations in the interaction of the cAMP cascade with normal substrates. The majority of Ca2+/calmodulin kinase activity (80–90%, depending on the homogenization procedure) was associated with S2 as revealed by phosphorylation of exogenous synapsin I. Two endogenous substrates for this kinase in P2 had molecular masses of approximately 45 and 87 kilodaltons. At least 11 substrates for the Ca2+/calmodulin-dependent kinase were detected in S2. The 45-kilodalton protein was a major substrate in this fraction too, as was pp37.

Notizen: Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.

Notizen: Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the “β-secretase” pathway, which generates β-amyloid (Aβ1–40/42; ∼4 kDa), and the “α-secretase” pathway, which generates a smaller fragment, the “p3” peptide (Aβ17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp1), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu11) at the N terminus, rather than Aβ(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPPα release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.

Notizen: Calsenilin, which was originally identified as a presenilin interacting protein, has since been shown to be involved in the processing of presenilin(s), the modulation of amyloid β-peptide (Aβ) levels and apoptosis. Subsequent to its original identification, calsenilin was shown to act as a downstream regulatory element antagonist modulator (and termed DREAM), as well as to interact with and modulate A-type potassium channels (and termed KChIP3). Calsenilin is primarily a cytoplasmic protein that must translocate to the nucleus to perform its function as a transcriptional repressor. This study was designed to determine the cellular events that modulate the translocation of calsenilin from the cytoplasm to the nucleus. The nuclear translocation of calsenilin was found to be enhanced following serum deprivation. A similar effect was observed when cells were treated with pharmacological agents that directly manipulate the levels of intracellular calcium (caffeine and the calcium ionophore A23187), suggesting that the increased levels of calsenilin in the nucleus are mediated by changes in intracellular calcium. A calsenilin mutant that was incapable of binding calcium retained the ability to translocate to the nucleus. Taken together, these findings indicate that the level of intracellular calcium can modulate the nuclear translocation of calsenilin and that this process does not involve the direct binding of calcium to calsenilin.

Notizen: Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or ‘shedding’ of the large APP ectodomain can occur via one of two competing pathways, the α- and β-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The α-secretase pathway cleaves within the amyloidogenic Aβ domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the α- and β-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of α-secretase-released, soluble APP (sAPPα). However, statin-induced stimulation of sAPPα release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPα secretion. These results suggest that statins may regulate α-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A ‘phospho-proteomic’ analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.

Notizen: The Alzheimer amyloid precursor protein (APP) is a phosphoprotein, and the phosphorylation state of APP at Ser655 can be regulated by protein kinase C, calcium/calmodulin-dependent protein kinase II, and okadaic acid-sensitive protein phosphatases. Other enzymes may also play a role at Ser655 of APP and, perhaps, at other residues.Signal transduction via protein phosphorylation regulates APP metabolism. In particular, APP processing via the nonamyloidogenic secretory cleavage pathway is increased following the activation of protein kinase C or the inactivation of okadaic acid-sensitive protein phosphatases.The mechanism(s) by which protein phosphorylation regulates APP secretory cleavage include (among others): substrate activation, substrate redistribution, protease activation and/or protease redistribution. Current experimental evidence will be discussed, addressing the relative importance of each of these possibilities and the implications for these events in the modulation of β/A4-amyloidogenesis.

Notizen: [Auszug] Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid β-amyloid (Aβ) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Aβ accumulation. Recent studies indicate that postmenopausal ...