Platelet-derived Growth Factor Induces the β-γ-Secretase-mediated Cleavage of Alzheimer's Amyloid Precursor Protein through a Src-Rac-dependent Pathway

Abstract

The ␤-amyloid peptide (A␤) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as ␤-and ␥-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APPGal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cellconditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the ␥-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases A␤ generation and that, in cells stably expressing APP, the generation of A␤ is strongly decreased by the Src tyrosine kinase inhibitor PP2. ␤-Amyloid (A␤)1 deposition in the so-called amyloid plaques is one of the main features of Alzheimer's pathology. ␤-Amyloid consists of ϳ4-kDa peptides derived from the proteolytic processing of a membrane protein named amyloid precursor protein (APP). This amyloidogenic processing is driven by two enzyme activities, ␤-site APP cleaving enzyme (BACE) and ␥-secretase. BACE cleaves APP at 28 residues from the boundary between the extracellular/intraluminal domain of APP and the transmembrane domain of the protein (for a review see Ref. 1), releasing a large soluble protein, including nearly all the extracellular/intraluminal part of APP, and a short transmembrane peptide, including the 99 C-terminal residues of APP. This transmembrane C99 stub is a substrate for the ␥-secretase activity, which cleaves it, in a presenilin-dependent fashion, within the membrane ␣-helix, giving rise to the A␤ peptide 40 -42 amino acids long and to a peptide named APP intracellular domain (AID), which includes the small C-terminal cytosolic domain of APP (for a review see Refs. 2 and 3).The functions of APP and its proteolytic processing are still unknown. However, although the functions of the APP ectodomain remain elusive, there is increasing evidence that its cytodomain is the center of a complex network of interactions with several proteins, involved in vesicle transport and in signal transduction. In fact, it was demonstrated that APP cytoplasmic domain interacts with kinesin light chain and contributes to vesicles transport (4), thus suggesting that APP cleavage could regulate the transport of vesicles in the axons. On the other hand, the APP cytodomain binds several PTB domain-containing pro-

The c-Abl Tyrosine Kinase Phosphorylates the Fe65 Adaptor Protein to Stimulate Fe65/Amyloid Precursor Protein Nuclear Signaling

“…Thus, these different findings are most probably the result of the different GAL4-APP fusions used in these two studies. Indeed, the earlier work of Gianni et al (38) was an elegant study aimed at understanding the mechanisms regulating APP cleavage and not APP/Fe65-mediated transcription as in our work. Nevertheless, we tested whether c-Abl⌬XB also stimulated reporter gene activity that was driven by an APP-GAL4 DNA binding domain fusion in which GAL4 DNA binding domain sequences were fused to the C terminus of APP (APP-GAL4).…”

“…As such, phosphorylation of APP Tyr-682 or Fe65 Tyr-547 by active c-Abl appears not to influence APP/Fe65 interactions in any overt manner. Thus, while the stimulatory effect of c-Abl⌬XB on APP/Fe65 transcription is mediated by Fe65 Tyr-547 phosphorylation, this does not involve any marked changes in binding of APP to Fe65. Platelet-derived growth factor has recently been shown to induce ␤,␥-secretase cleavage of APP and this was characterized by monitoring the transcriptional activity of an APP-GAL4 fusion gene similar to the ones we used here (38). In the course of this study, active c-Abl was shown to have no effect on APP-GAL4-mediated transcription (38).…”

“…Thus, while the stimulatory effect of c-Abl⌬XB on APP/Fe65 transcription is mediated by Fe65 Tyr-547 phosphorylation, this does not involve any marked changes in binding of APP to Fe65. Platelet-derived growth factor has recently been shown to induce ␤,␥-secretase cleavage of APP and this was characterized by monitoring the transcriptional activity of an APP-GAL4 fusion gene similar to the ones we used here (38). In the course of this study, active c-Abl was shown to have no effect on APP-GAL4-mediated transcription (38). However, the APP-GAL4 fusion used in this earlier work comprised full-length GAL4 (containing both its DNA binding and transactivation domains) such that while transcription of the reporter gene required APP cleavage, it did not require the transactivation capability of the APP C-terminal domain.…”

“…Full-length APP containing the GAL4 DNA binding domain at its C terminus was created by mutating residue 148 of the GAL4 sequence to a stop codon in plasmid APP-GAL4 (kind gift of Tomasso Russo). Plasmid APP-GAL4 encodes a chimeric protein comprising full-length GAL4 fused to the C terminus of APP (38). Mutation of GAL4 residue 148 truncates the GAL4 sequence so that it contains only the DNA binding domain.…”

RAC1 Inhibition Targets Amyloid Precursor Protein Processing by γ-Secretase and Decreases Aβ Production in Vitro and in Vivo

“…Interestingly, two different aspects of APP processing appear to be controlled by Rac1 and other small G proteins: ectodomain shedding (20), which is a prerequisite for ␤-␥-secretase cleavage, and the ␤-␥-secretase cleavage itself (21)(22)(23). In particular, overexpression of dominant negative (RacN17) or constitutively active (RacQL) mutants of Rac1 was demonstrated to inhibit or stimulate ␥-secretase-mediated APP processing (21), respectively, which suggests that Rac1 is crucial for the homeostasis of endogenous A␤ production.…”