Biomedical subjects

Steven P Gygi

Publications and source records attributed to Steven P Gygi.

At least 37 records · Page 2Linked to original sources

PubMed2005

Cdc28-dependent regulation of the Cdc5/Polo kinase.

Polo kinase is activated as cells enter mitosis and plays a central role in coordinating diverse mitotic events, yet the mechanisms leading to activation of Polo kinase are poorly understood . Work in Xenopus meiotic cell cycles has suggested that Polo kinase functions in a pathway that helps trigger activation of Cdk1 . However, studies in other organisms have suggested that activation of Polo kinase is dependent upon Cdk1 and therefore occurs downstream of Cdk1 activation . In this study, we have investigated the role of Cdk1 in the activation of budding yeast Polo kinase. The budding yeast homologs of Cdk1 and Polo kinase are referred to as Cdc28 and Cdc5. We show that signaling from Cdc28 is required to maintain Cdc5 activity in vivo. Furthermore, purified Cdc28 associated with the mitotic cyclin Clb2 is sufficient to activate purified Cdc5 in vitro. A single Cdc28 consensus phosphorylation site found at threonine 242 in the activation loop segment of Cdc5 is required for Cdc5 function in vivo and for kinase activity in vitro, whereas four other Cdc28 consensus sites are dispensable. Analysis of Cdc5 phosphorylation by mass spectrometry indicates that threonine 242 is phosphorylated in vivo. These results suggest that Cdc28 activates Cdc5 via phosphorylation of threonine 242.

Amino Acid Sequence↗

PubMed2005

mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events.

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling.

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.

Animals↗

PubMed2005

Cdk1-dependent regulation of the mitotic inhibitor Wee1.

The Wee1 kinase phosphorylates and inhibits cyclin-dependent kinase 1 (Cdk1), thereby delaying entry into mitosis until appropriate conditions have been met. An understanding of the mechanisms that regulate Wee1 should provide new insight into how cells make the decision to enter mitosis. We report here that Swe1, the budding-yeast homolog of Wee1, is directly regulated by Cdk1. Phosphorylation of Swe1 by Cdk1 activates Swe1 and is required for formation of a stable Swe1-Cdk1 complex that maintains Cdk1 in the inhibited state. Dephosphorylation of Cdk1 leads to further phosphorylation of Swe1 and release of Cdk1. Thus, Cdk1 both positively and negatively regulates its own inhibitor. Regulation of the Swe1-Cdk1 complex is likely to play a critical role in controlling the transition into mitosis.

CDC2 Protein Kinase↗

PubMed2005

A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

Cell Cycle Proteins↗

PubMed2005

Characterization of mouse spleen cells by subtractive proteomics.

Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45- and CD45+ cell populations. Using shotgun protein analysis we identified approximately 2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45- and not CD45+ cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45- cells. Characterization of the proteins exclusively identified in CD45- cells was performed using gene ontology terms via the Javascript GoMiner. The CD45- cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal.

Animals↗

PubMed2005

The Parkinson's disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis.

Mutations in the DJ-1 gene cause early-onset autosomal recessive Parkinson's disease (PD), although the role of DJ-1 in the degeneration of dopaminergic neurons is unresolved. Here we show that the major interacting-proteins with DJ-1 in dopaminergic neuronal cells are the nuclear proteins p54nrb and pyrimidine tract-binding protein-associated splicing factor (PSF), two multifunctional regulators of transcription and RNA metabolism. PD-associated DJ-1 mutants exhibit decreased nuclear distribution and increased mitochondrial localization, resulting in diminished co-localization with co-activator p54nrb and repressor PSF. Unlike pathogenic DJ-1 mutants, wild-type DJ-1 acts to inhibit the transcriptional silencing activity of the PSF. In addition, the transcriptional silencer PSF induces neuronal apoptosis, which can be reversed by wild-type DJ-1 but to a lesser extent by PD-associated DJ-1 mutants. DJ-1-specific small interfering RNA sensitizes cells to PSF-induced apoptosis. Both DJ-1 and p54nrb block oxidative stress and mutant alpha-synuclein-induced cell death. Thus, DJ-1 is a neuroprotective transcriptional co-activator that may act in concert with p54nrb and PSF to regulate the expression of a neuroprotective genetic program. Mutations that impair the transcriptional co-activator function of DJ-1 render dopaminergic neurons vulnerable to apoptosis and may contribute to the pathogenesis of PD.

Apoptosis↗

PubMed2005

The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle.

Proteasome activity is fine-tuned by associating the proteolytic core particle (CP) with stimulatory and inhibitory complexes. Although several mammalian regulatory complexes are known, knowledge of yeast proteasome regulators is limited to the 19-subunit regulatory particle (RP), which confers ubiquitin-dependence on proteasomes. Here we describe an alternative proteasome activator from Saccharomyces cerevisiae, Blm10. Synthetic interactions between blm10Delta and other mutations that impair proteasome function show that Blm10 functions together with proteasomes in vivo. This large, internally repetitive protein is found predominantly within hybrid Blm10-CP-RP complexes, representing a distinct pool of mature proteasomes. EM studies show that Blm10 has a highly elongated, curved structure. The near-circular profile of Blm10 adapts it to the end of the CP cylinder, where it is properly positioned to activate the CP by opening the axial channel into its proteolytic chamber.

DNA-Binding Proteins↗

PubMed2005

Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1.

Homeostatic mechanisms in mammals respond to hormones and nutrients to maintain blood glucose levels within a narrow range. Caloric restriction causes many changes in glucose metabolism and extends lifespan; however, how this metabolism is connected to the ageing process is largely unknown. We show here that the Sir2 homologue, SIRT1--which modulates ageing in several species--controls the gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator PGC-1alpha. A nutrient signalling response that is mediated by pyruvate induces SIRT1 protein in liver during fasting. We find that once SIRT1 is induced, it interacts with and deacetylates PGC-1alpha at specific lysine residues in an NAD(+)-dependent manner. SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC-1alpha, but does not regulate the effects of PGC-1alpha on mitochondrial genes. In addition, SIRT1 modulates the effects of PGC-1alpha repression of glycolytic genes in response to fasting and pyruvate. Thus, we have identified a molecular mechanism whereby SIRT1 functions in glucose homeostasis as a modulator of PGC-1alpha. These findings have strong implications for the basic pathways of energy homeostasis, diabetes and lifespan.

Acetylation↗

PubMed2005

Photochromic agents as tools for protein structure study: lapachenole is a photoaffinity ligand of cytochrome P450 3A4.

Cytochrome P450 3A4 is a drug-metabolizing enzyme of extraordinarily broad substrate specificity. This quality imparts upon the enzyme special importance in understanding its determinants of activity and substrate recognition. Limited successes in P450 3A4 active-site structure studies have been achieved by use of mechanism-based inactivators and photoaffinity ligands. We report here the potential of photochromic agents, compounds with the ability to undergo light-induced, reversible reactions, to be used as effective photoaffinity ligands. Four such compounds of the chromene family were shown by ultraviolet and visible spectroscopy to undergo photoinduced rearrangements to highly conjugated and reactive products in buffered aqueous solution. While some of these intermediates were very long-lived (>12 h, photoactivated lapachenole), others existed for milliseconds in their opened forms (precocene I and 2,2-dimethyl-5,6-benzo-2H-chromene) and were observed by laser flash photolysis. Each of the tricyclic structures studied rapidly underwent Michael addition reactions with the test nucleophile glutathione upon irradiation to form single conjugated products. The smaller precocene I reacted more extensively to form multiple products. These attributes of the chromenes inspired testing of their potential to label cytochrome P450 3A4 in a light-dependent fashion. Access to the protein active site by lapachenole was demonstrated with the molecule's ability to competitively inhibit P450 3A4-mediated oxidative metabolism of midazolam with an IC(50) value of 71 microM. This inhibition became irreversible upon irradiation of the enzyme-ligand complex with ultraviolet light. These results clearly demonstrate that chromenes are effective photoaffinity reagents for the cytochrome P450 superfamily of enzymes and probably other proteins as well.

Acetaminophen↗

PubMed2005

The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications.

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.

Animals↗

PubMed2005

Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors.

Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 ribosomal S6 kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological kinase inhibitors we uncovered a ERK-independent, protein kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.

Binding Sites↗

PubMed2005

A quantitative proteomic analysis of growth factor-induced compositional changes in lipid rafts of human smooth muscle cells.

Signals that promote proliferation and migration of smooth muscle cells (SMC) have been implicated in pathologic growth of hollow organs. Members of the platelet-derived growth factor (PDGF) family, potent mitogens and motility factors for SMC, have been shown to signal through cholesterol-enriched lipid rafts. We recently demonstrated that PDGF-stimulated DNA synthesis in urinary tract SMC was dependent on the integrity of lipid rafts. Despite its known ability to rapidly alter discrete proteins within rafts, the effect of PDGF on overall raft protein composition is unknown. In this study, we employed isotope coded affinity tag (ICAT) analysis to evaluate PDGF-induced protein changes in lipid rafts of primary culture human SMC. Following acute (i.e., 15 min) exposure of SMC to PDGF, 23 proteins increased in rafts >20%. In contrast, raft localization of only three proteins increased after 12 h of PDGF treatment. Among the proteins that increased at 15 min were the glycophosphatidylinositol-anchored proteins Thy-1, 5'-nucleotidase, and CD55, the cytoskeletal proteins actin, actinin, tropomyosin-3 and -4, and the endocytosis-related proteins clathrin and beta-adaptin. In addition, eight Rho family members were localized to rafts by ICAT analysis. Collectively, these observations suggest a role for lipid rafts in regulation of PDGF-stimulated changes in the cytoskeleton.

Cells, Cultured↗

PubMed2005

Proteomic insights into ubiquitin and ubiquitin-like proteins.

The dynamic and specific modification of cellular proteins by members of the ubiquitin protein family is a vital regulatory mechanism that lies at the heart of almost all biological processes. Because of both their pervasive and complex nature, these regulatory pathways have been the target of many recent proteomic studies. Such works have provided numerous insights. Through the use of various mass spectrometry techniques, affinity purification methods, and/or chemical probes, large lists have begun to be compiled for the multitude of substrates, interacting partners, and enzymatic components of these regulatory circuits. Furthermore, similar tools have provided many insights into functional aspects such as their mechanisms of substrate specificity and enzymatic activity. This review provides a summary of these recent proteomic works, along with comments on future directions of the field.

Humans↗

PubMed2005

Phosphorylation of ACAP1 by Akt regulates the stimulation-dependent recycling of integrin beta1 to control cell migration.

Components of intracellular signaling that mediate the stimulation-dependent recycling of integrins are being identified, but key transport effectors that are the ultimate downstream targets remain unknown. ACAP1 has been shown recently to function as a transport effector in the cargo sorting of transferrin receptor (TfR) that undergoes constitutive recycling. We now show that ACAP1 also participates in the regulated recycling of integrin beta1 to control cell migration. However, in contrast to TfR recycling, the role of ACAP1 in beta1 recycling requires its phosphorylation by Akt, which is, in turn, regulated by a canonical signaling pathway. Disrupting the activities of either ACAP1 or Akt, or their assembly with endosomal beta1, inhibits beta1 recycling and cell migration. These findings advance an understanding of how integrin recycling is achieved during cell migration, and also address a basic issue of how intracellular signaling can interface with transport to achieve regulated recycling.

ADP-Ribosylation Factor 6↗

PubMed2005

An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets.

With the recent exponential increase in protein phosphorylation sites identified by mass spectrometry, a unique opportunity has arisen to understand the motifs surrounding such sites. Here we present an algorithm designed to extract motifs from large data sets of naturally occurring phosphorylation sites. The methodology relies on the intrinsic alignment of phospho-residues and the extraction of motifs through iterative comparison to a dynamic statistical background. Results show the identification of dozens of novel and known phosphorylation motifs from recently published serine, threonine and tyrosine phosphorylation studies. When applied to a linguistic data set to test the versatility of the approach, the algorithm successfully extracted hundreds of language motifs. This method, in addition to shedding light on the consensus sequences of identified and as yet unidentified kinases and modular protein domains, may also eventually be used as a tool to determine potential phosphorylation sites in proteins of interest.

Algorithms↗

PubMed2005

Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics.

Mass-spectrometry-based proteomics has become an essential tool for the qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the ubiquitin system are well suited to proteomic studies. This review summarizes advances involving the identification of ubiquitinated proteins, the elucidation of ubiquitin-modification sites and the determination of polyubiquitin chain linkages, as well as offering a perspective on the application of emerging technologies for mechanistic and functional studies of protein ubiquitination.

Animals↗

PubMed2005

Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations.

Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.

Algorithms↗