Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication.

SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein-protein interactions.

Systematic genetic analysis with ordered arrays of yeast deletion mutants.

In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.

Cell signalling - the proteomics of it all.

A challenge for biomedical scientists today is to arrive at an understanding of cellular behavior on a global scale. The advent of DNA microarrays has greatly facilitated discovery of gene expression profiles associated with different cellular states. The problem of understanding cellular signaling at the level of the interacting proteins is in some ways more challenging. Ashman et al. discuss the current methods available for studying protein interactions on a global scale, as well as directions for the future. Technical hurdles exist at many stages, from the isolation of protein complexes, to the determination of their composition, to the software and databases needed to analyze the results of large-scale, high-throughput datasets. Ashman et al. suggest that, with advances in technology and cooperation among academia and industry, a global protein interaction map that underlies cellular behavior will emerge as an essential resource for basic and applied research.

Ubiquitin junction, what's your function?

A report on the Ubiquitin and Intracellular Protein Degradation FASEB summer conference, Saxtons River, USA, 23-28 June 2001.

AFM 4.0: a toolbox for DNA microarray analysis.

We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software.

MAPK specificity in the yeast pheromone response independent of transcriptional activation.

The mechanisms whereby different external cues stimulate the same mitogen-activated protein kinase (MAPK) cascade, yet trigger an appropriately distinct biological response, epitomize the conundrum of specificity in cell signaling. In yeast, shared upstream components of the mating pheromone and filamentous growth pathways activate two related MAPKs, Fus3 and Kss1, which in turn regulate programs of gene expression via the transcription factor Ste12. As fus3, but not kss1, strains are impaired for mating, Fus3 exhibits specificity for the pheromone response. To account for this specificity, it has been suggested that Fus3 physically occludes Kss1 from pheromone-activated signaling complexes, which are formed on the scaffold protein Ste5. However, we find that genome-wide expression profiles of pheromone-treated wild-type, fus3, and kss1 deletion strains are highly correlated for all induced genes and, further, that two catalytically inactive versions of Fus3 fail to abrogate the pheromone-induced transcriptional response. Consistently, Fus3 and Kss1 kinase activity is induced to an equivalent extent in pheromone-treated cells. In contrast, both in vivo and in an in vitro-reconstituted MAPK system, Fus3, but not Kss1, exhibits strong substrate selectivity toward Far1, a bifunctional protein required for polarization and G(1) arrest. This effect accounts for the failure to repress G(1)-S specific transcription in fus3 strains and, in part, explains the mating defect of such strains. MAPK specificity in the pheromone response evidently occurs primarily at the substrate level, as opposed to specific kinase activation by dedicated signaling complexes.

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

BACKGROUND: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete. RESULTS: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine. CONCLUSIONS: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.

The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27.

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.

Tyrosine-phosphorylated low density lipoprotein receptor-related protein 1 (Lrp1) associates with the adaptor protein SHC in SRC-transformed cells.

v-Src transforms fibroblasts in vitro and causes tumor formation in the animal by tyrosine phosphorylation of critical cellular substrates. Exactly how v-Src interacts with these substrates remains unknown. One of its substrates, the adaptor protein Shc, is thought to play a crucial role during cellular transformation by v-Src by linking v-Src to Ras. We used Shc proteins with mutations in either the phosphotyrosine binding (PTB) or Src homology 2 domain to determine that phosphorylation of Shc in v-Src-expressing cells depends on the presence of a functional PTB domain. We purified a 100-kDa Shc PTB-binding protein from Src-transformed cells that was identified as the beta chain of the low density lipoprotein receptor-related protein LRP1. LRP1 acts as an import receptor for a variety of proteins and is involved in clearance of the beta-amyloid precursor protein. This study shows that LRP1 is tyrosine-phosphorylated in v-Src-transformed cells and that tyrosine-phosphorylated LRP1 binds in vivo and in vitro to Shc. The association between Shc and LRP1 may provide a mechanism for recruitment of Shc to the plasma membrane where it is phosphorylated by v-Src. It is at the membrane that Shc is thought to be involved in Ras activation. These observations further suggest that LRP1 could function as a signaling receptor and may provide new avenues to investigate its possible role during embryonal development and the onset of Alzheimer's disease.

Regulation of cell cycle progression by Swe1p and Hog1p following hypertonic stress.

Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells. Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells. Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected. Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity. Thus, deletion of SWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity. Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation. Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay. However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p. These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.

Outbreak of tuberculosis associated with a church.

Investigation of an outbreak of tuberculosis (TB) in a West Midlands health district in 1999 revealed spread in an extended family network and to church contacts. Within the family four cases of smear positive TB, four cases of smear negative infection, and 14 cases requiring chemoprophylaxis were identified. One of the infectious cases visited a local church on two occasions, which resulted in a further 16 cases of infection including one case of tuberculous meningitis. DNA fingerprinting of isolates from five culture positive cases indicated that the same strain of Mycobacterium tuberculosis was responsible. This outbreak is a reminder that while outbreaks of TB usually arise within households or family networks, where close contact over extended periods provides more opportunity for exposure, community outbreaks of TB can occur after only causal contact.

Transcriptional regulation: Kamikaze activators.

Transcription factors are often targeted for rapid degradation by the ubiquitin-proteasome system. Recent evidence points to a correlation between the potency and instability of transcriptional activators, suggesting a possible direct role for ubiquitin-dependent proteolysis in transcriptional activation.

SCF(Met30)-mediated control of the transcriptional activator Met4 is required for the G(1)-S transition.

Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.

Proteolysis and the cell cycle: with this RING I do thee destroy.

The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins. Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits. The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCF(Met30 )complex.

Saccharomyces cerevisiae SCF(Met30) ubiquitin-protein ligase controls cell cycle function and sulfur amino acid metabolism. We report here that the SCF(Met30 )complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S-adenosylmethionine (AdoMet) increases. This AdoMet-induced Met4p degradation is dependent upon the 26S proteasome function. Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level. We provide evidence that the interaction between Met4p and the F-box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCF(Met30) complex. We also demonstrate that Met30p is a short-lived protein, which localizes within the nucleus. Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function. Thus Met4p appears to control its own degradation by regulating the amount of assembled SCF(Met30) ubiquitin ligase.

The fork'ed path to mitosis.

A concurrence of genomic, reverse genetic and biochemical approaches has cracked the decade-long enigma concerning the identity of the transcription factors that control gene expression at the G2/M transition in the budding yeast cell cycle.

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.