RESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes.
Assuntos
Núcleo Celular/química , Holoenzimas/química , Proteína Fosfatase 1/química , Receptores de Neuropeptídeo Y/química , Animais , Sítios de Ligação , Células COS , Núcleo Celular/genética , Chlorocebus aethiops/genética , Células HEK293 , Holoenzimas/genética , Humanos , Imunoprecipitação , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/genética , Receptores de Neuropeptídeo Y/genética , Especificidade por SubstratoRESUMO
Protein phosphatase 1 (PP1), a serine/threonine protein phosphatase, controls diverse key cellular events. PP1 catalytic subunits form complexes with a variety of interacting proteins that control its ability to dephosphorylate substrates. Here we show that the human mitotic kinesin-8, KIF18A, directly interacts with PP1γ through a conserved RVxF motif. Our phylogenetic analyses of the kinesins further uncovered the broad conservation of this interaction potential within the otherwise highly diverse motor-protein superfamily. This suggests an ancestral origin of PP1 recruitment to KIF18A and a strategic role in human mitotic cells.
Assuntos
Cinesinas/metabolismo , Mitose , Proteína Fosfatase 1/metabolismo , Sítios de Ligação , Células HeLa , Humanos , FilogeniaRESUMO
The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, ß and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Domínios de Homologia de srcRESUMO
Nasopharyngeal swabs are considered the preferential collection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. Less invasive and simpler alternative sampling procedures, such as saliva collection, are desirable. We compared saliva specimens and nasopharyngeal (NP) swabs with respect to sensitivity in detecting SARS-CoV-2. A nasopharyngeal and two saliva specimens (collected by spitting or oral swabbing) were obtained from >2500 individuals. All samples were tested by RT-qPCR, detecting RNA of SARS-CoV-2. The test sensitivity was compared on the two saliva collections with the nasopharyngeal specimen for all subjects and stratified by symptom status and viral load. Of the 2850 patients for whom all three samples were available, 105 were positive on NP swab, whereas 32 and 23 were also positive on saliva spitting and saliva swabbing samples, respectively. The sensitivity of the RT-qPCR to detect SARS-CoV-2 among NP-positive patients was 30.5% (95% CI, 1.9%-40.2%) for saliva spitting and 21.9% (95% CI, 14.4%-31.0%) for saliva swabbing. However, when focusing on subjects with medium to high viral load, sensitivity on saliva increased substantially: 93.9% (95% CI, 79.8%-99.3%) and 76.9% (95% CI, 56.4%-91.0%) for spitting and swabbing, respectively, regardless of symptomatic status. Our results suggest that saliva cannot readily replace nasopharyngeal sampling for SARS-CoV-2 diagnostics but may enable identification of the most contagious cases with medium to high viral loads.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Saliva/virologia , Manejo de Espécimes/métodos , Adulto , COVID-19/etiologia , Portador Sadio/virologia , Humanos , Nasofaringe/virologia , Estudos Prospectivos , Manejo de Espécimes/instrumentação , Carga ViralRESUMO
Protein phosphorylation appears to be a universal mechanism of protein regulation. Genomics has provided the means to compile inventories of protein phosphatases across a wide selection of organisms and this has supplied insights into the evolution of this group of enzymes. Protein phosphatases evolved independently several times yielding the groups we observe today. Starting from a core catalytic domain, phosphatases evolved by a series of gene duplication events and by adopting the use of regulatory subunits and/or fusion with novel functional modules or domains. Recent analyses also suggest that the serine/threonine specific enzymes are more ancient than the PTPs (protein tyrosine phosphatases). It is likely that the latter played a key role at the onset of metazoan evolution in conjunction with the tremendous expansion of tyrosine kinases and PTPs at this point. In the present review, we discuss the evolution of the PTPs, the serine/threonine specific PPP (phosphoprotein phosphatase) and PPM (metallo-dependent protein phosphatase) families and the more recently discovered phosphatases that utilize an aspartate-based catalytic mechanism. We will also highlight examples of convergent evolution and several phosphatases which are unique to plants.
Assuntos
Evolução Molecular , Fosfoproteínas Fosfatases/genética , Plantas/genética , Animais , Ativação Enzimática , Humanos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/metabolismo , Plantas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. RESULTS: We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIalpha, several nuclear helicases, NUP153 and the TRRAP complex. CONCLUSION: This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.
Assuntos
Cromatografia de Afinidade/métodos , Proteína Fosfatase 1/isolamento & purificação , Motivos de Aminoácidos , Animais , Sítios de Ligação , Glicogênio/isolamento & purificação , Células HeLa , Humanos , Microcistinas/química , Microcistinas/metabolismo , Músculo Esquelético/metabolismo , Proteína Fosfatase 1/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Coelhos , Sefarose/químicaRESUMO
The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Proteína Quinase Ativada por DNA/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
We have identified Cdc55, a regulatory B subunit of protein phosphatase 2A (PP2A), as an essential activating factor for stress gene transcription in Saccharomyces cerevisiae. The presence of PP2A-Cdc55 is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. We show that PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 during hyperosmolarity stress. PP2A-Cdc55 also enhances Msn2-dependent transactivation, required for extended chromatin recruitment of the transcription factor. We analyzed a possible direct regulatory role for PP2A-Cdc55 on the phosphorylation status of Msn2. Detailed mass spectrometric and genetic analysis of Msn2 showed that stress exposure causes immediate transient dephosphorylation of Msn2 which is not dependent on PP2A-Cdc55 activity. Furthermore, the Hog1 mitogen-activated protein kinase pathway activity is not influenced by PP2A-Cdc55. We therefore propose that the PP2A-Cdc55 phosphatase is not involved in cytosolic stress signal perception but is involved in a specific intranuclear mechanism to regulate Msn2 and Msn4 nuclear accumulation and chromatin association under stress conditions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Pressão Osmótica , Fosforilação , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Ativação Transcricional , Dedos de ZincoRESUMO
Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis.
Assuntos
RNA Helicases DEAD-box/metabolismo , Mitose/fisiologia , Proteína Fosfatase 1/metabolismo , Fuso Acromático/metabolismo , Ciclo Celular/fisiologia , RNA Helicases DEAD-box/genética , Humanos , Fosforilação , Proteína Fosfatase 1/genética , Fuso Acromático/genéticaRESUMO
The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic 'RVxF' motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.
RESUMO
A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1ß and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Far-Western Blotting , Northern Blotting , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Espectrometria de Massas , Proteômica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismoRESUMO
In yeast, glucose depletion elicits a quick response in the transcription of stress-related genes. The main transcriptional activator that orchestrates this response is Msn2, whose nuclear localization and DNA binding are negatively controlled by the cAMP-dependent protein kinase (PKA). Msn2 activation by sudden glucose depletion correlates with a fast but transient decrease in phosphorylation of several sites in its nuclear localization signal (NLS). Here we show that protein phosphatase 1 (PP1) is the direct antagonist of PKA-dependent phosphorylation at the Msn2 nuclear import domain and therefore a potential mediator of glucose starvation signals that target this transcription factor. Apart from PKA, the protein kinase Snf1 can also directly modify one of the Msn2 phosphorylation sites (S582) and thereby repress Msn2 function. Consequently, in snf1 mutants, rephosphorylation of the NLS happens to be much slower during prolonged starvation. Thus, a second, Reg1-dependent form of PP1 indirectly influences Msn2 functionality by modulating Snf1 kinase activation and repression. Different activities of PP1 are therefore involved in shaping induction and adaptation of the transcriptional stress response during acute glucose starvation.