Eric C. Chang

Cooperative interaction of S. pombe proteins required for mating and morphogenesis

We isolated two S. pombe genes, scd1 and scd2, that are required for normal morphology and mating. scd1 and scd2 are homologous to CDC24 and BEM1, respectively, of S. cerevisiae. Epistasis analyses indicate that scd2 and ras1 converge upon scd1, which, in turn, interacts with cdc42sp, a RHO-like GTPase. Studies with the yeast two-hybrid system indicate that scd2 forms complexes with both scd1 and cdc42sp. Furthermore, biochemical studies indicate that the interaction between scd1 and scd2 is direct. The yeast two-hybrid data further suggest that scd1, scd2, cdc42sp, and ras1, in its GTP-bound state, act cooperatively to form a protein complex.

Serum interleukin-6 as a marker of periprosthetic infection following total hip and knee arthroplasty.

BACKGROUND The erythrocyte sedimentation rate, the C-reactive protein serum level, and the white blood-cell count are routinely used to diagnose periprosthetic infection. In the present study, the diagnostic accuracy of the interleukin-6 serum level was compared with the accuracy of these standard tests for the evaluation of a group of patients who had had a total hip or total knee arthroplasty and were undergoing a reoperation for the treatment of an infection or another implant-related problem. METHODS A prospective, case-control study of fifty-eight patients who had had a total hip or knee replacement and were undergoing a reoperation because of an infection (seventeen patients) or another implant-related problem (forty-one patients) was conducted. The serum levels of interleukin-6 and C-reactive protein, the erythrocyte sedimentation rate, and the white blood-cell count were measured. The definitive diagnosis of an infection was determined on the basis of positive histopathological evidence of infection and growth of bacteria on culture of intraoperative specimens. Two-sample Wilcoxon rank-sum (Mann-Whitney) tests were used to determine the presence of a significant difference between patients with and without infection with regard to each laboratory value studied. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of each text were also calculated. RESULTS The serum interleukin-6 level, erythrocyte sedimentation rate, and C-reactive protein level were significantly higher in patients who had an infection than in those who did not, both when all patients were considered together and when the total hip arthroplasty and total knee arthroplasty groups were considered separately. With the numbers available, there was no significant difference with regard to the white blood-cell count between patients with and without infection. With a normal serum interleukin-6 level defined as <10 pg/mL, the serum interleukin-6 test had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 1.0, 0.95, 0.89, 1.0, and 97%, respectively. CONCLUSIONS An elevated serum interleukin-6 level correlated positively with the presence of periprosthetic infection in patients undergoing a reoperation at the site of a total hip or knee arthroplasty. The serum interleukin-6 level is valuable for the diagnosis of periprosthetic infection in patients who have had a total hip or total knee arthroplasty.

The eIF3 Interactome Reveals the Translasome, a Supercomplex Linking Protein Synthesis and Degradation Machineries

eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-beta Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-beta is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physically linked to facilitate efficient protein synthesis.

Schizosaccharomyces pombe Int6 and Ras Homologs Regulate Cell Division and Mitotic Fidelity via the Proteasome

Yin6 is a yeast homolog of Int6, which is implicated in tumorigenesis. We show that Yin6 binds to and regulates proteasome activity. Overexpression of Yin6 strengthens proteasome function while inactivation weakens and causes the accumulation of polyubiquitinated proteins including securin/Cut2 and cyclin/Cdc13. Yin6 regulates the proteasome by preferentially interacting with Rpn5, a conserved proteasome subunit, and affecting its localization/assembly. We showed previously that Yin6 cooperates with Ras1 to mediate chromosome segregation; here, we demonstrate that Ras1 similarly regulates the proteasome via Rpn5. In yeast, human Int6 binds Rpn5 and regulates its localization. We propose that human Int6, either alone or cooperatively with Ras, influences proteasome activities via Rpn5. Inactivating Int6 can lead to accumulation of mitotic regulators affecting cell division and mitotic fidelity.

Two Ras Pathways in Fission Yeast Are Differentially Regulated by Two Ras Guanine Nucleotide Exchange Factors

ABSTRACT How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity. Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs. Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating. So far there is only one established Ras1 GEF, Ste6. Paradoxically, ste6 null (ste6Δ) mutants are sterile but normal in cell morphology. This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway. We thereby characterized a potential GEF, Efc25. Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway. Like ras1Δ and scd1Δ, efc25Δ is synthetically lethal with a deletion in tea1, a critical element for cell polarity control. Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus. We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway. While ste6 expression is induced during mating, efc25 expression is constitutive. Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1. This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2. Reciprocally, Ste6 overexpression can block Scd1 activation. We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.

Recurrent ESR1 – CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers

Characterizing the genetic alterations leading to the more aggressive forms of estrogen receptor positive (ER+) breast cancers are of critical significance in breast cancer management. Here we identify recurrent rearrangements between estrogen receptor gene ESR1 and its neighbor CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal-B tumors, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170 positive tumors. These fusions encode N-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity, and enhanced xenograft tumor formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signaling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies

ABSTRACT Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.

Direct Binding and In Vivo Regulation of the Fission Yeast p21-Activated Kinase Shk1 by the SH3 Domain Protein Scd2

ABSTRACT The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.

Spatial Segregation of Ras Signaling—New Evidence from Fission Yeast

The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question—how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras.

ZOMES III: the interface between signalling and proteolysis

The ZOMES III meeting was organized by W. Dubiel, M. Naumann, D. Chamovitz and M. Glickman, and was held in Berlin, Germany, between 9 and 12 May, 2004. The meeting title refers to the fact that two of the protein complexes discussed, namely the COP9 signalosome and the 26S proteasome, end with the suffix ‘‐some’ ![][1] It is generally assumed that evolutionarily conserved protein domains participate in similar biochemical processes. Frequently, however, similar protein domains are found in the subunits of protein complexes that, at first glance, have no functions in common. For example, the ‘lid’ of the 26S proteasome regulates the degradation of proteins that are marked for proteolysis by polyubiquitylation (Glickman et al , 1998), the eukaryotic translation initiation factor 3 (eIF3) complex is essential for initiating messenger RNA (mRNA) translation (Asano et al , 1997) and the COP9 signalosome (CSN) was first shown to control photomorphogenesis in Arabidopsis (Chamovitz et al , 1996; Kwok et al , 1996) but has since been found to be central to many other signalling events (see below). At first glance, these three complexes do not seem to have anything in common; however, many of their subunits contain either a Proteasome–COP9–Initiation factor (PCI) or an Mpr1–Pad1–amino (N)‐terminus (MPN) domain. The presence of similar domains in complexes with apparently divergent functions challenges us to think more critically and creatively about their role in the cell, and about what advantages there might be to recruiting the same protein domain into functionally distinct protein machineries. For example, could similar molecular mechanisms govern fundamentally different cellular functions? Or could the related domains allow interaction with the same molecular targets, thereby facilitating the assembly of similar subunits into a complex? For the third time since 1999, around 100 scientists from all over the world gathered at the ZOMES III meeting to … [1]: /embed/graphic-1.gif