Edgar F. da Cruz e Silva

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.

Protein Phosphorylation and APP Metabolism

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimers amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimers disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed Aβ. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and Aβ production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimers amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimers disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed Aβ. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and Aβ production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.

Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.

Protein phosphatase 1 complexes modulate sperm motility and present novel targets for male infertility

Infertility is a growing concern in modern society, with 30% of cases being due to male factors, namely reduced sperm concentration, decreased motility and abnormal morphology. Sperm cells are highly compartmentalized, almost devoid of transcription and translation consequently processes such as protein phosphorylation provide a key general mechanism for regulating vital cellular functions, more so than for undifferentiated cells. Reversible protein phosphorylation is the principal mechanism regulating most physiological processes in eukaryotic cells. To date, hundreds of protein kinases have been identified, but significantly fewer phosphatases (PPs) are responsible for counteracting their action. This discrepancy can be explained in part by the mechanism used to control phosphatase activity, which is based on regulatory interacting proteins. This is particularly true for PP1, a major serine/threonine-PP, for which >200 interactors (PP1 interacting proteins-PIPs) have been indentified that control its activity, subcellular location and substrate specificity. For PP1, several isoforms have been described, among them PP1γ2, a testis/sperm-enriched PP1 isoform. Recent findings support our hypothesis that PP1γ2 is involved in the regulation of sperm motility. This review summarizes the known sperm-specific PP1-PIPs, involved in the acquisition of mammalian sperm motility. The complexes that PP1 routinely forms with different proteins are addressed and the role of PP1/A-kinase anchoring protein complexes in sperm motility is considered. Furthermore, the potential relevance of targeting PP1-PIPs complexes to infertility diagnostics and therapeutics as well as to male contraception is also discussed.

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.

Aβ promotes Alzheimer’s disease‐like cytoskeleton abnormalities with consequences to APP processing in neurons

J. Neurochem. (2010) 113, 761–771.

Alternatively Spliced Protein Variants as Potential Therapeutic Targets for Male Infertility and Contraception

Abstract: Mammalian sperm were previously shown to express the PP1γ2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1γ1 and PP1γ2 (the two known alternatively spliced variants of PP1γ). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two‐hybrid method. Among the various proteins detected, the most abundant interactors with PP1γ2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A‐T and NIPP1‐T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT‐PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A‐T and NIPP1‐T variants appear to bind PP1γ2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis‐specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1γ2, Nek2A‐T, and NIPP1‐T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.

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.

PP1 inhibition by Aβ peptide as a potential pathological mechanism in Alzheimer's disease

Abnormal protein phosphorylation has been associated with several neurodegenerative disorders, including Alzheimers disease (AD). Abeta is the toxic peptide that results from proteolytic cleavage of the Alzheimers amyloid precursor protein, a process where protein phosphatases are known to impact. The data presented here demonstrates that protein phosphatase 1 (PP1), an abundant neuronal serine/threonine-specific phosphatase highly enriched in dendritic spines, is specifically inhibited by Abeta peptides both in vitro and ex vivo. Indeed, the pathologically relevant Abeta(1-40) and Abeta(1-42) peptides, as well as Abeta(25-35), specifically inhibit PP1 with low micromolar potency, as compared to inactive controls and other disease related peptides (e.g. the prion related Pr(118-135) and Pr(106-126)). Interestingly, PP1 inhibition is increased by Abeta aggregation, indicating a possible direct neurotoxic effect of the aggregated peptide. PP1 involvement in processes like long-term depression, memory and learning, and synaptic plasticity, prompt us to suggest that PP1 may constitute an important physiological target for Abeta and, therefore, increased Abeta production and/or aggregation may lead to abnormal PP1 activity and likely contribute to the progressive neuropsychiatric AD condition. Thus, PP1 activity and levels constitute potential biomolecular candidates for diagnostics and therapeutics.

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.