The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Ubiquitin System

Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.

http://science.sciencemag.org/content/sci/274/5293/1672.full.pdf

Cancer Cell Cycles

A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.

/pdf/the-perseus-computational-platform-for-comprehensive-5a4p3g7pp4.pdf

The Perseus computational platform for comprehensive analysis of (prote)omics data.

The retinoblastoma protein and cell cycle control

Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.

/pdf/regulation-of-chromatin-by-histone-modifications-35b8poynlc.pdf

Regulation of chromatin by histone modifications

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1 alpha (PP1 alpha) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1 alpha as critical regulators of centrosome structure.

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities.

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C

Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.

The role of isoprenylation in membrane attachment of nuclear lamins A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.

Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome.

Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Doree, J. C. Labbe, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.

/pdf/expression-of-chicken-lamin-b2-in-escherichia-coli-2fvo5wmi0e.pdf

Erich A. Nigg

Papers

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C

The role of isoprenylation in membrane attachment of nuclear lamins A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties

Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome.

Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions.