Mark G. Goebl

SKP1 Connects Cell Cycle Regulators to the Ubiquitin Proteolysis Machinery through a Novel Motif, the F-Box

We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.

Identification, analysis, and prediction of protein ubiquitination sites

Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry, and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class‐balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half‐lives were significantly enriched in the number of predicted ubiquitination sites. Proteome‐wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory, and cancer‐associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease‐associatedmutations. UbPred is available at http://www.ubpred.org. Proteins 2010.

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein.

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

Cdc53p acts in concert with cdc4p and cdc34p to control the G1-to-S- phase transition and identifies a conserved family of proteins

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

Phosphorylation of nuclear MyoD is required for its rapid degradation.

ABSTRACT MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).

Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae.

Glycogen, a branched polymer of glucose, is a storage molecule whose accumulation is under rigorous nutritional control in many cells. We report the identification of two Saccharomyces cerevisiae genes, GLG1 and GLG2, whose products are implicated in the biogenesis of glycogen. These genes encode self-glucosylating proteins that in vitro can act as primers for the elongation reaction catalyzed by glycogen synthase. Over a region of 258 residues, the Glg proteins have 55% sequence identify to each other and approximately 33% identity to glycogenin, a mammalian protein postulated to have a role in the initiation of glycogen biosynthesis. Yeast cells defective in either GLG1 or GLG2 are similar to the wild type in their ability to accumulate glycogen. Disruption of both genes results in the inability of the cells to synthesize glycogen despite normal levels of glycogen synthase. These results suggest that a self-glucosylating protein is required for glycogen biosynthesis in a eukaryotic cell. The activation state of glycogen synthase in glg1 glg2 cells is suppressed, suggesting that the Glg proteins may additionally influence the phosphorylation state of glycogen synthase.

Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA

Although the entire DNA sequence of the yeast genome has been determined, the functions of nearly a third of the identified genes are unknown. Recently, we described a collection of mutants, each with a transposon‐tagged disruption in an essential gene in Saccharomyces cerevisiae. Identification of these essential genes and characterization of their mutant phenotypes should help assign functions to these thousands of novel genes, and since each mutation in our collection is physically marked by the uniform, unique DNA sequence of the transposable element, it should be possible to use the polymerase chain reaction (PCR) to amplify the DNA adjacent to the transposon. However, existing PCR methods include steps that make their use on a large scale cumbersome. In this report, we describe a semi‐random, two‐step PCR protocol, ST‐PCR. This method is simpler and more specific than current methods, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. Using this method, we have rapidly and easily identified the essential genes identified by several of our mutants. ©1997 John Wiley & Sons, Ltd.

The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.

Cutting Edge: Mouse Pellino-2 Modulates IL-1 and Lipopolysaccharide Signaling

Pellino is a Drosophila protein originally isolated in a two-hybrid screen for proteins interacting with the serine/threonine kinase, pelle. Although mammalian homologs have been identified in mouse and man, the function of pellino is as yet unknown. In this study, the cloning, expression pattern, and a preliminary characterization of mouse pellino-2 is described. These studies reveal that mouse pellino-2 is expressed during embryogenesis and in a tissue-restricted manner in the adult. IL-1 induces the association of mouse pellino-2 with the mouse pelle-like kinase/IL-1R-associated kinase protein, a mammalian homolog of pelle. Ectopic pellino-2 expression did not result in NF-κB activation. However, ectopic expression of a mouse pellino-2 antisense construct inhibited IL-1 or LPS-induced activation of NF-κB-dependent IL-8 promoter activity. Our data reveal that mouse pellino-2 is a tissue-restricted component of a signaling pathway that couples the mouse pelle-like kinase/IL-1R-associated kinase protein to IL-1- or LPS-dependent signaling.

Modulation of Tumor Necrosis Factor and Interleukin-1-dependent NF-κB Activity by mPLK/IRAK

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-κB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, themouse Pelle-likekinase/human interleukin-1receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-κB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-κB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-κB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-κB-dependent gene expression.