Sandra Rebelo

Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

BackgroundRetrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN) occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimers disease (AD). The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimers amyloid precursor protein (APP), events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s) in APP that regulate its sorting to the TGN have not been characterized.ResultsThrough the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life.ConclusionsWe reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to the TGN or lysosomes. Significantly, the data are consistent with known interactions involving the retromer, SorLA and APP. Further, these findings add to our understanding of APP targeting and potentially contribute to our knowledge of sporadic AD pathogenesis representing putative new targets for AD therapeutic strategies.

Identification of the human testis protein phosphatase 1 interactome

Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

S655 phosphorylation enhances APP secretory traffic.

Cellular protein phosphorylation regulates proteolytic processing of the Alzheimer’s Amyloid Precursor Protein (APP). This appears to occur both indirectly and directly via APP phosphorylation at residues within cytoplasmic motifs related to targeting and protein–protein interactions. The sorting signal 653YTSI656 comprises the S655 residue that can be phosphorylated by PKC, particularly in mature APP molecules. The YTSI domain has been associated with APP internalization and Golgi polarized sorting, but no functional significance has been attributed to S655 phosphorylation thus far. Using APP695-GFP S655 phosphomutants we show that S655 phosphorylation is a signal that positively modulates APP secretory traffic. The phosphomimicking and dephosphomimicking S655 mutants exhibited contrasting Golgi dynamics, which correlated with differential Golgi vesicular exit and secretory cleavage to sAPP. The role of S655 phosphorylation in APP trafficking at sorting stations, such as the Golgi, its contribution toward cytoprotective alpha sAPP production, and implications for Alzheimer’s disease are discussed.

Enhanced generation of Alzheimer’s amyloid‐β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer’s amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid‐β (Aβ) fragments of APP, although identification of the PKC substrate phospho‐state‐sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum‐free medium is associated with several phenomena, namely: (i) PKCα down‐regulation; (ii) PKCε up‐regulation; (iii) accumulation of APP and/or APP carboxyl‐terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid‐lowering strategies based on modulating PKC activity.

Signal transduction therapeutics: relevance for Alzheimer's disease.

It is now widely accepted that abnormal processing of the Alzheimer’s amyloid precursor protein (APP) can contribute significantly to Alzheimer’s disease (AD). APP can be processed proteolytically to give rise to several fragments, including toxic β-amyloid (Aβ) fragments that are subsequently deposited as amyloid plaques in brains of AD patients. Data from several groups have revealed that APP processing can be regulated by phosphorylation and phosphorylation-dependent events. Consequently, the key players controlling such signal transduction cascades, the protein kinases and phosphatases, as well as their corresponding regulatory proteins, take on added importance. By characterizing how altered cell signaling might contribute to APP processing, one can identify potential targets for signal transduction therapeutics. Here, we review APP phosphorylation and phosphorylation-dependent events in APP processing, with particular focus on phosphatases that impact on APP processing, and their binding and regulatory proteins. Particular attention is given to protein phosphatase 1 (PP1), as it seems to have a central role not only in the regulation of APP cleavage events but also in the molecular control of neurotransmission and in age-related memory deterioration. The development of specific drugs targeting protein phosphatase binding proteins would constitute potential therapeutic agents with a high degree of specificity. The identification of such targets provides novel therapeutic avenues for normal aging and for neurodegenerative conditions such as AD.

Protein phosphatase 1 is a key player in nuclear events.

Reversible protein phosphorylation at serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is among the major regulatory mechanism in eukaryotic cells. The eukaryotic genome encodes many protein kinases and protein phosphatases. However, the localization, activity and specificity towards phosphatase substrates are dictated by a large array of phosphatase binding and regulatory subunits. For protein phosphatase 1 (PP1) more than 200 binding subunits have been described. The various PP1 isoforms and the binding subunits can be located throughout the cell, including in the nucleus. It follows that several nuclear specific PP1 binding proteins (PIPs) have been described and these will be discussed. Among them are PNUTS (phosphatase 1 nuclear targeting subunit), NIPP1 (nuclear inhibitor of PP1) and CREB (cAMP-responsive element-binding protein), which have all been associated with transcription. In fact PP1 can associate with transcription factors fulfilling an important regulatory function, in this respect it can bind to Hox11, human factor C1 (HCF1) and myocyte enhancer factor-2 (MEF2). PP1 also regulates cell cycle progression and centrosome maturation and splitting, again by binding to specific regulatory proteins. Moreover, PP1 together with other protein phosphatases control the entry into mitosis by regulating the activity of mitotic kinases. Thus, PP1, its binding proteins and/or the phosphorylation states of both, directly control a vast array of cell nucleus associated functions, many of which are starting to be unraveled.

Tyrosine 687 Phosphorylated Alzheimer’s Amyloid Precursor Protein Is Retained Intracellularly and Exhibits a Decreased Turnover Rate

Tyrosine 687 (Y687) of the Alzheimer’s amyloid precursor protein (APP) was shown to be phosphorylated in the brains of Alzheimer’s disease patients. This residue lies within a typical endocytosis consensus motif commonly found in molecules with receptor functions, strongly suggesting a potential role for APP in signal transduction. Consequently, the work here described addressed how phosphorylation of Y687 may be affecting APP in terms of its proteolytic cleavage and subcellular distribution. Our data show that the APP mutant mimicking constitutive dephosphorylation of Y687 had a faster turnover rate, both in terms of maturation and metabolism, when compared to Wt-APP-GFP and even more so when compared to the mutant mimicking constitutive phosphorylation. Thus, the mutant mimicking constitutively phosphorylated Y687 had a much higher t1/2 and was significantly retained both in the ER and TGN. Additionally, this mutant was not incorporated into visible vesicular structures, with a concomitant dramatic decrease in Aβ production. Our findings point to the direct phosphorylation of APP on Y687 as an important regulatory mechanism in terms of determining the subcellular localization of APP and modulating its processing via different proteolytic pathways.

The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.

A Model System to Study Intracellular Trafficking and Processing of the Alzheimer's Amyloid Precursor Protein

The occurrence of consensus phosphorylation sites in the intracellular domain of the Alzheimer’s amyloid precursor protein (APP), coupled with observations of their in vivo phosphorylation, prompted several workers to investigate the effects that phosphorylation of such sites could have on APP metabolism and subsequent Aβ production. However, hitherto all attempts to dissect the role played by such phosphorylation events failed to reveal substantial effects. Having decided to revisit this problem, our new approach was based on the following vectors: (1) site-directed mutagenesis of the target amino acids to mimic a specific phosphorylation state, (2) expression of wild-type and mutant APP-GFP (green fluorescent protein) fusion proteins for ease of visualization, (3) controlled low level expression to avoid ‘flooding’ cellular pathways, and (4) the use of cycloheximide to inhibit de novo protein synthesis. Using this method we were able to detect specific differences in APP processing that were correlated with the mimicked phosphorylation state of several phosphorylation sites. New combined methodologies, like the one described here, allow for the detailed analysis of key control points in the cellular metabolism of specific proteins that are central to neurodegenerative diseases and may be under the control of specific posttranslational modifications, such as reversible phosphorylation.

Isoform Specific Amyloid- β Protein Precursor Metabolism

Alzheimers amyloid-beta protein precursor (AbetaPP) can occur in different isoforms, among them AbetaPP(751), which is the most abundant isoform in non-neuronal tissues, and AbetaPP(695), often referred to as the neuronal isoform. However, few isoform-specific roles have been addressed. In the work here described, AbetaPP isoforms, both endogenous and as cDNA fusions with green fluorescent protein (GFP), were used to permit isoform-specific monitoring in terms of intracellular processing and targeting. Differences were particularly marked in the turnover rates of the immature isoforms, with AbetaPP(751) having a faster turnover rate than AbetaPP(695) (0.8 h and 1.2 h respectively for endogenous proteins and 1.1 h and 2.3 h for transfected proteins). Hence, AbetaPP(751) matures faster. Additionally, AbetaPP(751) responded to both okadaic acid (OA) and phorbol 12-myristate 13-acetate (PMA), as determined by sAbetaPP production, with PMA inducing a more robust response. For the AbetaPP(695) isoform, however, although PMA produced a strong response, OA failed to elicit such an induction in sAbetaPP production, implicating isoform specificity in phosphorylation regulated events. In conclusion, it seems that the AbetaPP(695) isoform is processed/metabolized at a slower rate and responds differently to OA when compared to the AbetaPP(751) isoform. The relevance of isoform-specific processing in relation to Alzheimers disease needs to be further investigated, given the predominance of the AbetaPP(695) isoform in neuronal tissues and isoform-specific alterations in expression levels associated with the pathology.