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PubMed2006

Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner.

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.

Animals↗
PubMed2006

Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species.

Animals↗
PubMed2005

Magnitude of the hydrophobic effect at central versus peripheral sites in protein-protein interfaces.

Hydrophobic interactions are essential for stabilizing protein-protein complexes, whose interfaces generally consist of a central cluster of hot spot residues surrounded by less important peripheral residues. According to the O-ring hypothesis, a condition for high affinity binding is solvent exclusion from interacting residues. This hypothesis predicts that the hydrophobicity at the center is significantly greater than at the periphery, which we estimated at 21 cal mol(-1) A(-2). To measure the hydrophobicity at the center, structures of an antigen-antibody complex where a buried phenylalanine was replaced by smaller hydrophobic residues were determined. By correlating structural changes with binding free energies, we estimate the hydrophobicity at this central site to be 46 cal mol(-1) A(-2), twice that at the periphery. This context dependence of the hydrophobic effect explains the clustering of hot spots at interface centers and has implications for hot spot prediction and the design of small molecule inhibitors.

Animals↗
PubMed2006

Endosomal and non-endosomal functions of ESCRT proteins.

The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.

Animals↗
PubMed2002

Regulation and targets of receptor tyrosine kinases.

Ligand-mediated activation of receptor tyrosine kinases (RTKs) results in autophosphorylation of both the receptor catalytic domain and noncatalytic regions of the cytoplasmic domain. Catalytic domain phosphorylation leads to activation and potentiation of receptor kinase activity. Noncatalytic domain phosphorylation creates docking sites for downstream cytoplasmic targets, which bind to specific receptor phosphotyrosine residues. Downstream signaling pathways are constructed in a modular fashion. In addition to SH2 and PTB (phosphotyrosine binding) domains, downstream signal proteins also contain domains that recognize other protein and phospholipid motifs. The arrangement and re-arrangement of various combinations of modular domains in different signaling proteins (combinatorial use) has allowed for the creation of complex signaling networks and pathways. In addition to performing catalytic functions, signaling proteins serve as scaffolds for the assembly of multiprotein signaling complexes, as adaptors, as transcription factors and as signal pathway regulators. Recent results show that the juxtamembrane region of Eph receptors is important in receptor autoregulation. Mutations in the juxtamembrane region of several RTKs have been shown to play a role in oncogenesis. It is likely that dysregulation of other modular components of signaling pathways also plays a role in oncogenic transformation.

Cell Communication↗
PubMed2003

The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis.

The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. Here we link the CSN to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, we show that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes similar defects as mutations in CSN or SCF(Ago) subunits: extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation.

Animals↗
PubMed2004

Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans.

The ESCRT-I, -II, and -III protein complexes function to create multivesicular bodies (MVBs) for sorting of proteins destined for the lysosome or vacuole. Prior studies with Saccharomyces cerevisiae have shown that the ESCRT-III protein Snf7p interacts with the MVB pathway protein Bro1p as well as its homolog Rim20p. Rim20p has no role in MVB formation, but functions in the Rim101p pH-response pathway; Rim20p interacts with transcription factor Rim101p and is required for the activation of Rim101p by C-terminal proteolytic cleavage. We report here that ESCRT-III proteins Snf7p and Vps20p as well as all ESCRT-I and -II proteins are required for Rim101p proteolytic activation in S. cerevisiae. Mutational analysis indicates that the Rim20p N-terminal region interacts with Snf7p, and an insertion in the Rim20p "Bro1 domain" abolishes this interaction, as determined with two-hybrid assays. Disruption of the MVB pathway through mutations affecting non-ESCRT proteins does not impair Rim101p processing. The relationship between the MVB pathway and Rim101p pathway is conserved in Candida albicans, because mutations in four ESCRT subunit genes abolish alkaline pH-induced filamentation, a phenotype previously seen for rim101 and rim20 mutants. The defect is suppressed by expression of C-terminally truncated Rim101-405p, as expected for mutations that block Rim101p proteolytic activation. These results indicate that the ESCRT complexes govern a specific signal transduction pathway and suggest that the MVB pathway may provide a signal that regulates pH-responsive transcription.

Candida albicans↗
PubMed2003

Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms.

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase.

COP9 Signalosome Complex↗
PubMed2005

The emerging role of the COP9 signalosome in cancer.

In the last several years, multiple lines of evidence have suggested that the COP9 signalosome (CSN) plays a significant role in the regulation of multiple cancers and could be an attractive target for therapeutic intervention. First, the CSN plays a key role in the regulation of Cullin-containing ubiquitin E3 ligases that are central mediators of a variety of cellular functions essential during cancer progression. Second, several studies suggest that the individual subunits of the CSN, particularly CSN5, might regulate oncogenic and tumor suppressive functions independently of, or coordinately with, the CSN holocomplex. Thus, deregulation of CSN subunit function can have a dramatic effect on diverse cellular functions, including the maintenance of DNA fidelity, cell cycle control, DNA repair, angiogenesis, and microenvironmental homeostasis that are critical for tumor development. Additionally, clinical studies have suggested that the expression or localization of some CSN subunits correlate to disease progression or clinical outcome in a variety of tumor types. Although the study of CSN function in relation to tumor progression is in its infancy, this review will address current studies in relation to cancer initiation, progression, and potential for therapeutic intervention.

Animals↗
PubMed2002

COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster.

The COP9 signalosome (CSN) is an essential eight-subunit repressor of light-regulated development in Arabidopsis. This complex has also been identified in animals, though its developmental role remains obscure. CSN subunits have been implicated in various cellular processes, suggesting a possible role for the CSN as an integrator of multiple signaling pathways. In order to elucidate the function of the CSN in animals, a Drosophila model system has previously been established. Gel-filtration analysis with antibodies against CSN subunits 4, 5 and 7 revealed that these proteins act as a complex in Drosophila that is similar in size to the plant and mammalian complexes. Null mutations in either one of two subunits, CSN4 or CSN5, are larval lethal. Successful embryogenesis appears to be a consequence of maternal contribution of the complex. Biochemical analysis indicates that the different subunits are found in both CSN-dependent and CSN-independent forms, and that these forms are differentially affected by the mutations. Phenotypic characterization of these two mutants indicates that they show both shared and unique phenotypes, which suggest specific roles for each subunit. Both mutants have defective oocyte and embryo patterning, and defects in response to DNA damage, while csn5 mutants develop melanotic tumors and csn4 mutants have phenotypes reminiscent of defects in ecdysone signaling.

Animals↗
PubMed2000

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry.

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.

Amino Acid Sequence↗
PubMed2005

Expression profiles of two human APH-1 genes and their roles in formation of presenilin complexes.

APH-1 is a polytopic membrane protein that functions as a component of presenilin-gamma-secretase complexes. Two homologous genes of APH-1 exist in humans, APH-1a and APH-1b, and alternative splicing of the former generates two isoforms, APH-1aS and APH-1aL. We performed semi-quantitative reverse transcription-PCR analysis to investigate mRNA expression of these three APH-1 forms in human cell lines and tissues. We found that both APH-1a and APH-1b were expressed in almost all tissues, and that APH-1aS was 1.5-3 times more abundantly expressed than APH-1aL. We examined the effect of small interfering RNA-mediated knock down of APH-1a or APH-1b on APH-1 mRNA expression and presenilin complex protein expression. We found that knock down of APH-1a, but not APH-1b, resulted in impaired maturation of nicastrin and reduced expression of presenilin 1, presenilin 2, and PEN-2 proteins. These findings indicate that APH-1a plays an essential role in the formation of presenilin-gamma-secretase complexes.

Cell Line↗
PubMed2006

Formation of signal transduction complexes during immobile phase of NGFR movements.

Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of NGF-receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF-receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF-receptor complexes in the immobile phase indicating signal transduction occurs during this phase. Thus, NGF signaling is performed through a repetitive random process to induce transient formation of signaling complexes.

Animals↗
PubMed2006

Focal adhesions: what's new inside.

The cytoplasmic side of focal adhesions is comprised of large molecular complexes that link transmembrane receptors, such as integrins, to the actin cytoskeleton and mediate signals modulating cell attachment, migration, proliferation, differentiation, and gene expression. These complexes are heterogeneous and dynamic structures that are apparent targets of regulatory signals that control the function of focal adhesions. Recent studies using genetic approaches in invertebrate and vertebrate systems have begun to reveal the structure and function of these complexes in vivo.

Animals↗
PubMed2006

Visualization of transient encounter complexes in protein-protein association.

Kinetic data on a number of protein-protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex. Site-directed mutagenesis and brownian dynamics simulations have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality or the presence of a short-range, non-specific attractive potential. Here, using paramagnetic relaxation enhancement, we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein-protein complex between the amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein-protein complexes.

Bacterial Proteins↗
PubMed2004

Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos.

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

Amino Acid Sequence↗