Showing papers by "Steven P. Gygi published in 2009"

Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution

Abstract: To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.

SDH5, a Gene Required for Flavination of Succinate Dehydrogenase, Is Mutated in Paraganglioma

Abstract: Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline loss-of-function mutations in the human SDH5 gene, located on chromosome 11q13.1, segregate with disease in a family with hereditary paraganglioma, a neuroendocrine tumor previously linked to mutations in genes encoding SDH subunits. Thus, a mitochondrial proteomics analysis in yeast has led to the discovery of a human tumor susceptibility gene.

Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex.

Abstract: Brown adipose cells are specialized to dissipate chemical energy in the form of heat, as a physiological defence against cold and obesity. PRDM16 (PR domain containing 16) is a 140 kDa zinc finger protein that robustly induces brown fat determination and differentiation. Recent data suggests that brown fat cells arise in vivo from a Myf5-positive, myoblastic lineage by the action of PRDM16 (ref. 3); however, the molecular mechanisms responsible for this developmental switch is unclear. Here we show that PRDM16 forms a transcriptional complex with the active form of C/EBP-beta (also known as LAP), acting as a critical molecular unit that controls the cell fate switch from myoblastic precursors to brown fat cells. Forced expression of PRDM16 and C/EBP-beta is sufficient to induce a fully functional brown fat program in naive fibroblastic cells, including skin fibroblasts from mouse and man. Transplantation of fibroblasts expressing these two factors into mice gives rise to an ectopic fat pad with the morphological and biochemical characteristics of brown fat. Like endogenous brown fat, this synthetic brown fat tissue acts as a sink for glucose uptake, as determined by positron emission tomography with fluorodeoxyglucose. These data indicate that the PRDM16-C/EBP-beta complex initiates brown fat formation from myoblastic precursors, and may provide opportunities for the development of new therapeutics for obesity and type-2 diabetes.

During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Abstract: Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

Abstract: Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics

Abstract: β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the β-secretase responsible for generating the amyloid-β protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few β-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative β-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.

Chaperone-mediated pathway of proteasome regulatory particle assembly

Abstract: The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber called the core particle (CP) where degradation takes place. The CP associates with the RP at either one or both ends. The RP can be further subdivided into the base and lid. Two studies from Finley and colleagues elucidate the pathway of RP assembly. They report the identification of three molecular chaperones that assist in the assembly of the RP. Assembly of the base proceeds through a complex consisting of five proteins, called BP1, which functions as an intermediate in the process. These studies show that RP assembly is a highly orchestrated process. The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber, called the core particle (CP), where degradation takes place. Here, and in a complimentary study, the pathway of RP assembly is elucidated, demonstrating that RP assembly is a highly orchestrated process. The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin–protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded1. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP–CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases–the Rpt proteins–and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study2 that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins.

Abstract: Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions.

Oxygen-Regulated β2-Adrenergic Receptor Hydroxylation by EGLN3 and Ubiquitylation by pVHL

Abstract: Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although beta-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the beta(2)-adrenergic receptor (beta(2)AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the beta(2)AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with beta(2)AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the beta(2)AR to serve as an endogenous beta(2)AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous beta(2)AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.

Hexameric assembly of the proteasomal ATPases is templated through their C termini

Abstract: The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber called the core particle (CP) where degradation takes place. The CP associates with the RP at either one or both ends. The RP can be further subdivided into the base and lid. Two studies from Finley and colleagues elucidate the pathway of RP assembly. They report the identification of three molecular chaperones that assist in the assembly of the RP. Assembly of the base proceeds through a complex consisting of five proteins, called BP1, which functions as an intermediate in the process. These studies show that RP assembly is a highly orchestrated process. The proteasome is a large proteolytic machine that degrades ubiquitin-tagged proteins. Substrates are recognized and unfolded by the regulatory particle (RP) and translocated into a central proteolytic chamber, called the core particle (CP), where degradation takes place. Here, and in a complimentary study, the pathway of RP assembly is elucidated, demonstrating that RP assembly is a highly orchestrated process. Substrates of the proteasome are recognized and unfolded by the regulatory particle, and then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-6, is situated within the base subassembly of the regulatory particle1. The ATPase ring sits atop the CP, with the Rpt carboxy termini inserted into pockets in the CP2,3,4,5,6. Here we identify a previously unknown function of the Rpt proteins in proteasome biogenesis through deleting the C-terminal residue from each Rpt in the yeast Saccharomyces cerevisiae. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.

TAK1 is an essential regulator of BMP signalling in cartilage

Abstract: TGFβ activated kinase 1 (TAK1), a member of the MAPKKK family, controls diverse functions ranging from innate and adaptive immune system activation to vascular development and apoptosis. To analyse the in vivo function of TAK1 in cartilage, we generated mice with a conditional deletion of Tak1 driven by the collagen 2 promoter. Tak1col2 mice displayed severe chondrodysplasia with runting, impaired formation of secondary centres of ossification, and joint abnormalities including elbow dislocation and tarsal fusion. This phenotype resembled that of bone morphogenetic protein receptor (BMPR)1 and Gdf5-deficient mice. BMPR signalling was markedly impaired in TAK1-deficient chondrocytes as evidenced by reduced expression of known BMP target genes as well as reduced phosphorylation of Smad1/5/8 and p38/Jnk/Erk MAP kinases. TAK1 mediates Smad1 phosphorylation at C-terminal serine residues. These findings provide the first in vivo evidence in a mammalian system that TAK1 is required for BMP signalling and functions as an upstream activating kinase for Smad1/5/8 in addition to its known role in regulating MAP kinase pathways. Our experiments reveal an essential role for TAK1 in the morphogenesis, growth, and maintenance of cartilage.

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Abstract: Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Abstract: Ubiquitin (Ub)–protein conjugates formed by purified ring-finger or U-box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST-fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non-forked Ub conjugates.

A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry

Abstract: Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This approach concurrently measured up to 90 site-specific peptide phosphorylation rates, reporting a diagnostic fingerprint for activated kinase pathways. We applied this unique kinome-activity profiling strategy in a variety of cellular settings, including mitogen stimulation, cell cycle, pharmacological inhibition of pathways, and to a panel of breast cancer cell lines. Finally, we identified the source of activity for a peptide (derived from a PI3K regulatory subunit) from our library. This peptide substrate demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a novel Src family kinase site in vivo.

A conserved CCCH-type zinc finger protein regulates mRNA nuclear adenylation and export.

Abstract: Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.

Methods, compositions and kits for high throughput kinase activity screening using mass spectrometry and stable isotopes

Abstract: A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. Half of these peptides immediately or rapidly showed robust and site-specific phosphorylation after incubation with serum-starved HEK293 cell Iysate. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. Activating kinase pathways through insulin or EGF stimulation reproducibly altered the phosphorylation rates of peptides derived from known pathway protein substrates. While examining cell-cycle-specific activities with the panel, a peptide derived from phosphoinositide 3 -kinase regulatory subunit demonstrated mitotic and tyrosine-specifϊc phosphorylation, which was confirmed to be a Src kinase site in vivo. The kinome activity profiling strategy was successfully applied with lysates of each of: cells manipulated by various combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.