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On a daily basis, planktonic organisms migrate vertically and thus experience widely varying conditions in their physico-chemical environment. In the Gulf of Finland, these changes are larger than values predicted by climate change scenarios predicted for the next century (up to 0.5 units in pH and 5°C in temperature). In this work, we are interested in how temporal variations in physico-chemical characteristics of the water column on a daily and weekly scale influence oxidative stress level and antioxidant responses in the planktonic copepod of the genus Acartia. Responses were determined from samples collected during a two-week field survey in the western Gulf of Finland, Baltic Sea. Our results showed that GST (Glutathione-S-transferase) enzyme activity increased in the surface waters between Weeks I and II, indicating antioxidant defense mechanism activation. This is most likely due to elevating temperature, pH, and dissolved oxygen observed between these two weeks. During Week II also GSSG (oxidized glutathione) was detected, indicating that copepods responded to stressor(s) in the environment. Our results suggest that Acartia copepods seem fairly tolerant to weekly fluctuations in environmental conditions in coastal and estuarine areas, in terms of antioxidant defense and oxidative stress. This could be directly connected to a very efficient glutathione cycling system acting as antioxidant defense system for neutralizing ROS and avoiding elevated levels of LPX.

Cysteine cathepsins normally function in the lysosomal degradation system where they are critical for the maintenance of cellular homeostasis and the MHC II immune response, and have been found to have major roles in several diseases and in tumor progression. Selective visualization of individual protease activity within a complex proteome is of major importance to establish their roles in both normal and tumor cells, thereby facilitating our understanding of the regulation of proteolytic networks. A generally accepted means to monitor protease activity is the use of small molecule substrates and activity-based probes. However, there are eleven human cysteine cathepsins, with a few of them displaying overlapping substrate specificity, making the development of small molecules that selectively target a single cathepsin very challenging. Here, we utilized HyCoSuL, a positional scanning substrate approach, to develop a highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231. Use of this probe enabled us to distinguish the activity of cathepsin L from that of other cathepsins, particularly cathepsin B, which is abundant and ubiquitously expressed in normal and transformed cell types. We found that cathepsin L localization in MDA-MB-231 cells greatly overlaps with that of cathepsin B, however, several cathepsin L-rich lysosomes lacked cathepsin B activity. Overall, these studies demonstrate that HyCoSuL-derived small molecule probes are valuable tools to image cathepsin L activity in living cells. This approach thus enables evaluation of cathepsin L function in tumorigenesis and is applicable to other cysteine cathepsins.

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood-brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1-eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO's effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival. Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32-46. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful., Competing Interests: CONFLICTS OF INTEREST None.

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

The heterogeneous group of diseases collectively termed cancer results not just from aberrant cellular proliferation but also from a lack of accompanying homeostatic cell death. Indeed, cancer cells regularly acquire resistance to programmed cell death, or apoptosis, which not only supports cancer progression but also leads to resistance to therapeutic agents. Thus, various approaches have been undertaken in order to induce apoptosis in tumor cells for therapeutic purposes. Here, we will focus our discussion on agents that directly affect the apoptotic machinery itself rather than on drugs that induce apoptosis in tumor cells indirectly, such as by DNA damage or kinase dependency inhibition. As the roles of the Bcl-2 family have been extensively studied and reviewed recently, we will focus in this review specifically on the inhibitor of apoptosis protein (IAP) family. IAPs are a disparate group of proteins that all contain a baculovirus IAP repeat domain, which is important for the inhibition of apoptosis in some, but not all, family members. We describe each of the family members with respect to their structural and functional similarities and differences and their respective roles in cancer. Finally, we also review the current state of IAPs as targets for anti-cancer therapeutics and discuss the current clinical state of IAP antagonists., Competing Interests: Competing interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

High-throughput screening (HTS) strategies and protocols have undergone significant development in the last decade. It is now possible to screen hundreds of thousands of compounds, each exploring multiple biological phenotypes and parameters, against various cell lines or model systems in a single setting. However, given the vast amount of data such studies generate, the fact that they use multiple reagents, and are often technician-intensive, questions have been raised about the variability, reliability and reproducibility of HTS results. Assessments of the impact of the multiple factors in HTS studies could arguably lead to more compelling insights into the robustness of the results of a particular screen, as well as the overall quality of the study. We leveraged classical, yet highly flexible, analysis of variance (ANOVA)-based linear models to explore how different factors contribute to the variation observed in a screening study of four different melanoma cell lines and 120 drugs over nine dosages studied in two independent academic laboratories. We find that factors such as plate effects, appropriate dosing ranges, and to a lesser extent, the laboratory performing the screen, are significant predictors of variation in drug responses across the cell lines. Further, we show that when sources of variation are quantified and controlled for, they contextualize claims of inconsistencies and reveal the overall quality of the HTS studies performed at each participating laboratory. In the context of the broader screening study, we show that our analysis can also elucidate the robust effects of drugs, even those within specific cell lines.

Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α., Competing Interests: The authors have declared that no competing interests exist.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Extracellular matrix, via its receptors the integrins, has emerged as a crucial factor in cancer development. The α2β1 integrin is a major collagen receptor that is widely expressed and known to promote cell migration and control tissue homeostasis. Growing evidence suggests that it can be a key pathway in cancer. Recent studies have shown that α2β1 integrin is a regulator of cancer metastasis either by promoting or inhibiting the dissemination process of cancer cells. The α2β1 integrin signaling can also enhance tumor angiogenesis. Emerging evidence supports a role for α2β1 integrin in cancer chemoresistance especially in hematological malignancies originating from the T cell lineage. In addition, α2β1 integrin has been associated with cancer stem cells. In this review, we will discuss the complex role of α2β1 integrin in these processes. Collagen is a major matrix protein of the tumor microenvironment and thus, understanding how α2β1 integrin regulates cancer pathogenesis is likely to lead to new therapeutic approaches and agents for cancer treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A key aim of systems biology is the reconstruction of molecular networks. We do not yet, however, have networks that integrate information from all datasets available for a particular clinical condition. This is in part due to the limited scalability, in terms of required computational time and power, of existing algorithms. Network reconstruction methods should also be scalable in the sense of allowing scientists from different backgrounds to efficiently integrate additional data. We present a network model of acute myeloid leukemia (AML). In the current version (AML 2.1), we have used gene expression data (both microarray and RNA-seq) from 5 different studies comprising a total of 771 AML samples and a protein-protein interactions dataset. Our scalable network reconstruction method is in part based on the well-known property of gene expression correlation among interacting molecules. The difficulty of distinguishing between direct and indirect interactions is addressed by optimizing the coefficient of variation of gene expression, using a validated gold-standard dataset of direct interactions. Computational time is much reduced compared to other network reconstruction methods. A key feature is the study of the reproducibility of interactions found in independent clinical datasets. An analysis of the most significant clusters, and of the network properties (intraset efficiency, degree, betweenness centrality, and PageRank) of common AML mutations demonstrated the biological significance of the network. A statistical analysis of the response of blast cells from 11 AML patients to a library of kinase inhibitors provided an experimental validation of the network. A combination of network and experimental data identified CDK1, CDK2, CDK4, and CDK6 and other kinases as potential therapeutic targets in AML.

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

We performed laboratory experiments to investi-gate whether the synthesis of the antioxidants α-tocopherol (vitamin E) and β-carotene in phytoplankton depends on changes in abiotic factors. Cultures of Nodularia spumigena, Phaeodactylum tricornutum, Skeletonema costatum, Dunaliella tertiolecta, Prorocentrum cordatum, and Rhodomonas salina were incubated at different tempe-ratures, photon flux densities and salinities for 48 h. We found that abiotic stress, within natural ecological ranges, affects the synthesis of the two antioxidants in different ways in different species. In most cases antioxidant production was stimulated by increased abiotic stress. In P. tricornutum KAC 37 and D. tertiolecta SCCAP K-0591, both good producers of this compound, α-tocopherol accumulation was negatively affected by environmentally induced higher photosystem II efficiency (Fv /Fm ). On the other hand, β-carotene accumulation was positively affected by higher Fv /Fm in N. spumigena KAC 7, P. tricornutum KAC 37, D. tertiolecta SCCAP K-0591 and R. salina SCCAP K-0294. These different patterns in the synthesis of the two compounds may be explained by their different locations and functions in the cell. While α-tocopherol is heavily involved in the protection of prevention of lipid peroxidation in membranes, β-carotene performs immediate photo-oxidative protection in the antennae complex of photosystem II. Overall, our results suggest a high variability in the antioxidant pool of natural aquatic ecosystems, which can be subject to short-term temperature, photon flux density and salinity fluctuations. The antioxidant levels in natural phytoplankton communities depend on species composition, the physiological condition of the species, and their respective strategies to deal with reactive oxygen species. Since α-tocopherol and β-carotene, as well as many other nonenzymatic antioxidants, are exclusively produced by photo-synthetic organisms, and are required by higher trophic levels through dietary intake, regime shifts in the phytoplankton as a result of large-scale environmental changes, such as climate change, may have serious consequences for aquatic food webs., (© 2014 Phycological Society of America.)

Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180(S1250L) mutant, but not wild type Dock180(WT), protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-inducing activity in transformed, but not in normal, cells. As with most anticancer agents, however, its clinical use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that they do so in a caspase-8-dependent manner. We further show that the compounds have no cytotoxicity as single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the most efficacious in binding to X-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2 degradation. Our studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell lines.

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure-activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of novel, potent antagonists of the inhibitor of apoptosis proteins (IAPs) were synthesized in a highly convergent and rapid fashion (≤6 steps) using the Ugi four-component reaction as the key step, thus enabling rapid optimization of binding potency. These IAP antagonists compete with caspases 3, 7, and 9 for inhibition by X chromosome-linked IAP (XIAP) and bind strongly (nanomolar binding constants) to several crucial members of the IAP family of cancer pro-survival proteins to promote apoptosis, with a particularly unique selectivity for melanoma IAP (ML-IAP). Experiments in cell culture revealed powerful cancer cell growth inhibitory activity in multiple (breast, ovarian, and prostate) cell lines with single agent toxicity at low nanomolar levels against SKOV-3 human ovarian carcinoma cells. Administration of the compounds to human foreskin fibroblast cells revealed no general toxicity to normal cells. Furthermore, computational modeling was performed, revealing key contacts between the IAP proteins and antagonists, suggesting a structural basis for the observed potency.

This study investigated stock-specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin-O-deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable-isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post-smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock-specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable-isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea., (© 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.)

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.

Resistance to apoptosis and chemotherapy is a hallmark of cancer cells, and it is a critical factor in cancer recurrence and patient relapse. Extracellular matrix (ECM) via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy. Several studies over the last decade have demonstrated that ECM/integrin signaling provides a survival advantage to various cancer cell types against numerous chemotherapeutic drugs and against antibody therapy. In this paper, we will discuss the major findings on how ECM/integrin signaling protects tumor cells from drug-induced apoptosis. We will also discuss the potential role of ECM in malignant T-cell survival and in cancer stem cell resistance. Understanding how integrins and their signaling partners promote tumor cell survival and chemoresistance will likely lead to the development of new therapeutic strategies and agents for cancer treatment.

Two hallmarks of glioblastoma multiforme, the most common malignant brain cancer in humans, are aggressive growth and the ability of single glioma cells to disperse throughout the brain. These characteristics render tumors resistant to current therapies and account for the poor prognosis of patients. Although it is known that oncogenic signaling caused by overexpression of genes such as PDGFRA is responsible for robust glioma growth and cell infiltration, the mechanisms underlying glioblastoma malignancy remain largely elusive. Here, we report that PDGFRα signaling in glioblastomas leads to Src-dependent phosphorylation of the guanine nucleotide exchange factor Dock180 at tyrosine 1811 (Dock180(Y1811)) that results in activation of the GTPase Rac1 and subsequent cell growth and invasion. In human glioma cells, knockdown of Dock180 and reversion with an RNAi-resistant Dock180(Y1811F) abrogated, whereas an RNAi-resistant Dock180(WT) rescued, PDGFRα-promoted glioma growth, survival, and invasion. Phosphorylation of Dock180(Y1811) enhanced its association with CrkII and p130(Cas), causing activation of Rac1 and consequent cell motility. Dock180 also associated with PDGFRα to promote cell migration. Finally, phosphorylated Dock180(Y1811) was detected in clinical samples of gliomas and various types of human cancers, and coexpression of phosphorylated Dock180(Y1811), phosphorylated Src(Y418), and PDGFRα was predictive of extremely poor prognosis of patients with gliomas. Taken together, our findings provide insight into PDGFRα-stimulated gliomagenesis and suggest that phosphorylated Dock180(Y1811) contributes to activation of Rac1 in human cancers with PDGFRA amplification.

Background: The focal adhesion protein p130Cas (Cas) activates multiple intracellular signaling pathways upon integrin or growth factor receptor ligation. Full-length Cas frequently promotes cell survival and migration, while its C-terminal fragment (Cas-CT) produced upon intracellular proteolysis is known to induce apoptosis in some circumstances. Here, we have studied the putative role of Cas in regulating cell survival and death pathways upon proteasome inhibition., Results: We found that Cas-/- mouse embryonic fibroblasts (MEFs), as well as empty vector-transfected Cas-/- MEFs (Cas-/- (EV)) are significantly resistant to cell death induced by proteasome inhibitors, such as MG132 and Bortezomib. As expected, wild-type MEFs (WT) and Cas-/- MEFs reconstituted with full-length Cas (Cas-FL) were sensitive to MG132- and Bortezomib-induced apoptosis that involved activation of a caspase-cascade, including Caspase-8. Cas-CT generation was not required for MG132-induced cell death, since expression of cleavage-resistant Cas mutants effectively increased sensitivity of Cas-/- MEFs to MG132. At the present time, the domains in Cas and the downstream pathways that are required for mediating cell death induced by proteasome inhibitors remain unknown. Interestingly, however, MG132 or Bortezomib treatment resulted in activation of autophagy in cells that lacked Cas, but not in cells that expressed Cas. Furthermore, autophagy was found to play a protective role in Cas-deficient cells, as inhibition of autophagy either by chemical or genetic means enhanced MG132-induced apoptosis in Cas-/- (EV) cells, but not in Cas-FL cells. Lack of Cas also contributed to resistance to the DNA-damaging agent Doxorubicin, which coincided with Doxorubicin-induced autophagy in Cas-/- (EV) cells. Thus, Cas may have a regulatory role in cell death signaling in response to multiple different stimuli. The mechanisms by which Cas inhibits induction of autophagy and affects cell death pathways are currently being investigated., Conclusion: Our study demonstrates that Cas is required for apoptosis that is induced by proteasome inhibition, and potentially by other death stimuli. We additionally show that Cas may promote such apoptosis, at least partially, by inhibiting autophagy. This is the first demonstration of Cas being involved in the regulation of autophagy, adding to the previous findings by others linking focal adhesion components to the process of autophagy., (© 2011 Zhao and Vuori; licensee BioMed Central Ltd.)

We report the systematic rational design and synthesis of new monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Characterization of compounds in vitro (including 9i; ML101) led to the determination of key structural requirements for BIR2 binding affinity. Compounds 9h and 9j sensitized TRAIL-resistant breast cancer cells to apoptotic cell death, highlighting the value of these probe compounds as tools to investigate the biology of XIAP., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS) strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be "weeded out" in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC) was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries.

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.

Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility.

Caspase-8 has a well-defined canonical role as an apical protease of the extrinsic apoptosis pathway. Evidence is growing, however, that the protein has numerous other nonapoptotic functions. We have previously shown that caspase-8 is required for efficient adhesion-induced activation of the extracellular signal-regulated kinase (Erk)-1/2 pathway. We now show that caspase-8 is also necessary for the efficient activation of downstream events associated with epidermal growth factor (EGF) signaling. This promotion of EGF-induced Erk1/2 activation is independent of the proteolytic activity of caspase-8 and can be recapitulated using only the pro-domains of the protein. In addition, we identify specific residues within the caspase-8 "RXDLL motif" that are essential for Erk pathway activation. Furthermore, these residues are also involved in forming a complex with the tyrosine kinase Src. Caspase-8 null cells and cells reconstituted with caspase-8 harboring point mutations of these critical amino acids also show defective EGF-induced migration as compared with cells reconstituted with the wild-type protein. In sum, we provide the first evidence for caspase-8 as an essential component of growth factor signaling and suggest that this may be due to its association with Src. As the EGF/Src pathway activity has been shown to promote oncogenic events, our findings that caspase-8 is necessary for these activities may help explain why it is rarely deleted or silenced in tumors.

An oxidative enol ether rearrangement was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies demonstrated potent integrin CD11b/CD18 antagonistic effects.

Dock1 (also known as Dock180) is a prototypical member of a new family of atypical Rho GTPase activators. Genetic studies in Drosophila and Caenorhabditis elegans have demonstrated that Dock1 orthologues in these organisms have a crucial role in activating Rac GTPase signaling. We generated mutant alleles of the closely related Dock1 and Dock5 genes to study their function in mammals. We report that while Dock5 is dispensable for normal mouse embryogenesis, Dock1 has an essential role in embryonic development. A dramatic reduction of all skeletal muscle tissues is observed in Dock1-null embryos. Mechanistically, this embryonic defect is attributed to a strong deficiency in myoblast fusion, which is detectable both in vitro and in vivo. Furthermore, we have uncovered a contribution of Dock5 toward myofiber development. These studies identify Dock1 and Dock5 as critical regulators of the fusion step during primary myogenesis in mammals and demonstrate that a specific component of the myoblast fusion machinery identified in Drosophila plays an evolutionarily conserved role in higher vertebrates.

Neuroblastomas are extremely aggressive, although heterogeneous, cancers with a poor prognosis upon metastasis. Some evidence has suggested a correlative silencing of caspase-8 with MYCN amplification in neuroblastoma. A prognostic effect of this silencing, however, has been disputed. We report here hitherto undescribed roles for caspase-8 in the modulation of cell adhesion and subsequent activation of the Erk signaling pathway. Re-expression of caspase-8 in neuroblastoma cells lacking endogenous caspase-8 expression was found to promote cell adhesion to extracellular matrix and to activate adhesion-dependent signaling pathways, such as the Erk kinase cascade. This function of caspase-8 occurred irrespective of its proteolytic activity. Additionally, a pool of caspase-8 was shown to co-localize with the Src tyrosine kinase at the cellular periphery. Furthermore, our studies showed that caspase-8 forms a physical protein complex with Src via its death effector domains (DED) and maintains the complex in a detergent-soluble fraction. We also show that the DEDs of caspase-8 alone are necessary and sufficient to recreate the adhesive and biochemical phenotypes observed with the full-length protein, suggesting that caspase-8 may exert these effects via its association with Src. This protein complex association of caspase-8 and Src, and concomitant downstream signaling events, may help reconcile why a potential tumor suppressor such as caspase-8 is rarely absent in cancers.

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in many human tumors and mediates processes such as cell proliferation, survival, adhesion, and motility. The natural product, wortmannin, has been widely used to study the functional consequences of PI3K inhibition in both normal and transformed cells in culture but is not a suitable cancer chemotherapeutic agent due to stability and toxicity issues. PX-866, an improved wortmannin analogue, displays significant antitumor activity in xenograft models. Here, we directly compare PX-866 and wortmannin in human cancer cell lines cultured in monolayer or as three-dimensional spheroids. Both PI3K inhibitors failed to inhibit monolayer cell growth at concentrations up to 100 nmol/L but strongly suppressed spheroid growth at low nanomolar concentrations, with PX-866 showing greater potency than wortmannin. Relative to wortmannin, PX-866 treatment results in a more sustained loss of Akt phosphorylation, suggesting that the increased potency of PX-866 is related to a more durable inhibition of PI3K signaling. PX-866 and wortmannin both inhibit spheroid growth without causing cytotoxicity, similar to known cytostatic agents, such as rapamycin. PX-866 also inhibits cancer cell motility at subnanomolar concentrations. These findings suggest that the antitumor activities of PX-866 stem from prolonged inhibition of the PI3K pathway and inhibition of cell motility. In addition, we propose that the use of three-dimensional tumor models is more predictive of in vivo growth inhibition by PI3K inhibitors in cancer cell lines lacking phosphatase and tensin homologue activity or expression.

HGF, the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, which are processes that play a crucial role in cell motility during normal development and malignant transformation. We and others have shown earlier that the adapter protein CrkII and its associated proteins positively regulate cell migratory events in response to both haptotactic and chemotactic stimuli, including HGF. Here, we demonstrate for the first time that phosphorylation of CrkII serves as a negative feedback loop to regulate motile responses upon Met stimulation. Thus, we found that the treatment of cells with HGF induces tyrosine phosphorylation of CrkII at Y221, which in turn results in inhibition of CrkII signaling via formation of an intramolecular pY221-SH2-domain interaction. Accordingly, expression of a mutant form of CrkII, CrkII-Y221F, which is resistant to phosphorylation at this negative regulatory site, enhanced Met-induced cell motility. Furthermore, we demonstrate here that the Met-induced CrkII phosphorylation depends on the Abl tyrosine kinase activity. As a corollary, we found that Abl inhibitors, such as the STI571 compound, significantly enhanced Met-induced cell motility, but failed to do so in cells that expressed the CrkII-Y221F mutant protein. Taken together, these results demonstrate that the Abl tyrosine kinase functions as a negative regulator of Met-induced cell migration, and that it does so by inducing CrkII phosphorylation at the site Y221.

Rho GTPase activation, which is mediated by guanine nucleotide exchange factors (GEFs), is tightly regulated in time and space. Although Rho GTPases have a significant role in many biological events, they are best known for their ability to restructure the actin cytoskeleton profoundly through the activation of specific downstream effectors. Two distinct families of GEFs for Rho GTPases have been reported so far, based on the features of their catalytic domains: firstly, the classical GEFs, which contain a Dbl homology-pleckstrin homology domain module with GEF activity, and secondly, the Dock180-related GEFs, which contain a Dock homology region-2 domain that catalyzes guanine nucleotide exchange on Rho GTPases. Recent exciting data suggest key roles for the DHR-2 domain-containing GEFs in a wide variety of fundamentally important biological functions, including cell migration, phagocytosis of apoptotic cells, myoblast fusion and neuronal polarization.

Objective: To explore the cost of the statutory inpatient rehabilitation system in Finland and its impact on the functional and work capacity of patients with early rheumatoid arthritis (RA)., Methods: In the Finnish Rheumatoid Arthritis Combination-Therapy trial (FIN-RACo), 195 patients with recent-onset RA, 162 of them available for the work force, were randomly assigned to two different drug treatment strategies for 2 years. Otherwise, the patients received routine multidisciplinary care and, if their functional or work capacity was endangered, were referred to inpatient rehabilitation. After a 5-year follow-up, data on rehabilitation, sick leave, and RA-related disability pensions were obtained from official registers., Results: Of the 162 patients, 49 (30%) underwent inpatient rehabilitation at an average cost of EURO5400. The rehabilitated patients more often worked in white-collar jobs and had more pain and a worse Health Assessment Questionnaire (HAQ) score (1.0 vs. 0.78; p = 0.01) at baseline. Their HAQ scores remained higher throughout follow-up (p<0.001); no change appeared over inpatient periods [mean 0.01; 95% confidence interval (CI) -0.13 to 0.16]. No independent impact of rehabilitation on the HAQ score emerged in an adjusted generalized estimating equations (GEE) model (p = 0.55). Nor did any improvement in work capacity appear: average lost productivity (human capital approach) per patient-year was EURO10 155 (95% CI 6994-14 196) before and EUR 12 839 (95% CI 8589-19 139) after the start of rehabilitation., Conclusion: For patients with recent-onset RA, the Finnish statutory inpatient rehabilitation system had no positive impact on either functional or work capacity during the first few years, despite its considerable cost.

We report on the design, synthesis, and evaluation of a series of furanyl-salicyl-nitroxide derivatives as effective chemical probes for second-site screening against phosphotyrosine phosphatases (PTPs) using NMR-based techniques. The compounds have been tested against a panel of PTPs to assess their ability to inhibit a broad spectrum of these phosphatases. The utility of the derived compounds is illustrated with the phosphatase YopH, a bacterial toxin from Yersinia pestis. Novel chemical fragments were identified during an NMR-based screen for compounds that are capable of binding on the surface of YopH in regions adjacent the catalytic site in the presence of the spin-labeled compounds. Our data demonstrate the value of the derived chemical probes for NMR-based second-site screening in PTPs.

The aim of this study was to evaluate the effectiveness of IL-1 inhibition in rheumatic disease using real-life, observational methods. We analyzed data from 47 patients collected from the national ROB-FIN for rheumatic disease. Commonly used, validated measures of efficacy and adverse effects were documented and analyzed. The series contains 47/1,135 patients (mean age 47+/-11 years, range 25-73, females 83%) on anakinra of whom 39 patients suffered from rheumatoid arthritis (RA), two presented with psoriatic arthritis, and four with juvenile RA. At 3 months (26/40), 46% reached American College of Rheumatology (ACR) 20 and 27% ACR 50. In patients naive to biological drugs, the response rate at 3 months was 60% for ACR 20 and 20% for ACR 50. At follow-up of the total series, ACR responses at 6 and 12 months were 69/56% for ACR 20 and 23/22% for ACR 50. These data give room for IL-1 suppression when treating patient with rheumatic disease. Careful selection of patients, together with combining anakinra with disease-modifying antirheumatic drugs, perhaps adds effectiveness. For treating clinicians in Finland, these results are encouraging, as reimbursed treatment alternatives for patients refractory to all other therapies are still few.

Objective: To study the associations of tumor necrosis factor (TNF) a, b and c microsatellite markers with 1) the clinical disease activity and 2) the induction of remissions in patients with early rheumatoid arthritis (RA) treated with two treatment strategies., Methods: In the FIN-RACo (FINnish Rheumatoid Arthritis Combination therapy) trial of two years, 195 patients with recent-onset RA were randomly assigned to receive either a combination (COMBI) (sulphasalazine, methotrexate, hydroxychloroquine, and prednisolone) or a single (SINGLE) (initially sulphasalazine with or without prednisolone) disease modifying antirheumatic drug (DMARD) therapy. TNF a, b and c microsatellite and HLA-DRB1 typings were carried out in 165 (79 COMBI; 86 SINGLE) study completers., Results: At baseline the 28 joint disease activity scores (DAS28) of the patients positive for TNFa2, a13 or b1 microsatellite markers were significantly higher than in the other patients. In the SINGLE patients the DAS28 improved comparably in patients with (n = 31) or without (n = 53) the TNFb1 marker (NS), while the DAS28 of the TNFb1-positive COMBI patients (n = 22) improved significantly more than that of the TNFb1-negative cases (n = 57) (p = 0.014). Respective 31.8% (7/22) and 28.1% (16/57) of the COMBI patients with or without TNFb1 allele achieved remission at one year. The corresponding figure in SINGLE patients were 0% (0/31) and 20.8% (11/53) (p = 0.006). At two years the remission frequencies in the TNFb1+/TNFb1- patients in the COMBI and SINGLE were 50.0%/38.6% and 9.7%/22.6%, respectively., Conclusion: Early TNFb1+ RA patients have more active disease but respond more favourably to COMBI treatment than the patients without this microsatellite allele. The finding may be of clinical relevance for the choice of DMARDs in early RA.

A method based on Taylor series expansion for estimation of location parameters and variance components of non-linear mixed effects models was considered. An attractive property of the method is the opportunity for an easily implemented algorithm. Estimation of non-linear mixed effects models can be done by common methods for linear mixed effects models, and thus existing programs can be used after small modifications. The applicability of this algorithm in animal breeding was studied with simulation using a Gompertz function growth model in pigs. Two growth data sets were analyzed: a full set containing observations from the entire growing period, and a truncated time trajectory set containing animals slaughtered prematurely, which is common in pig breeding. The results from the 50 simulation replicates with full data set indicate that the linearization approach was capable of estimating the original parameters satisfactorily. However, estimation of the parameters related to adult weight becomes unstable in the case of a truncated data set.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling.

Protein-protein interactions between SHEP and Cas proteins influence cellular signaling through tyrosine kinases, as well as integrin-mediated signaling, and may be linked to antiestrogen resistance. Data from past studies suggests that association between SHEP and Cas proteins is critical for these cellular effects. In this study, the interacting domains of each protein were co-expressed in bacteria and a soluble stable complex was purified. Deuterium exchange mass spectrometry was used to define regions that are buried when SHEP1 is in complex with Cas. The results reveal four segments in SHEP1 that are highly protected, including a region (residues 619-640) that contains a key residue, tyrosine 635, required for association with Cas. This region is predominately hydrophilic, yet remains protected from solvent in the complex.

The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form., (Copyright 2005 John Wiley & Sons, Ltd.)

Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

Integrin-mediated cell adhesion induces activation of the EGF receptor tyrosine kinase independently of the soluble growth factor ligand. EGFR activation is instrumental for subsequent activation of additional signaling pathways in adherent cells, including the Ras-MAP kinase pathway and the phosphatidylinositol 3-kinase/Akt pathway. We demonstrate here that integrin-dependent EGFR activation is also essential for adhesion-induced formation of actin stress fibers, focal adhesion localization and tyrosine phosphorylation of the adapter protein paxillin, as well as transcriptional activation of the serum response factor. All these events are known to be mediated by the small GTPase RhoA. EGFR activity was not found to regulate the activity status of RhoA, however. Instead, we found that EGFR activity is required for integrin-induced phosphorylation of cofilin. Cofilin is an actin-binding protein, which, when unphosphorylated, stimulates depolymerization and severing of actin filaments. Thus, in the absence of the kinase activity of the EGFR, cofilin remains dephosphorylated and depolymerizes actin filaments, rendering cells unable to respond to RhoA signaling. These studies demonstrate adhesion-dependent regulation of cofilin phosphorylation, and identify a novel role for EGFR in integrin signaling.

The evolutionarily conserved DOCK180 protein has an indispensable role in cell migration by functioning as an exchange factor for Rac GTPase via its DOCK homology region (DHR)-2 domain. We report here that the conserved DHR-1 domain also has an important signalling role. A form of DOCK180 that lacks DHR-1 fails to promote cell migration, although it is capable of inducing Rac GTP-loading. The DHR-1 domain interacts with PtdIns(3,4,5)P(3) in vitro and in vivo, and mediates the DOCK180 signalling complex localization at sites of PtdIns(3,4,5)P(3) accumulation in the cell's leading edge. A form of DOCK180 in which the DHR-1 domain has been replaced by a canonical PtdIns(3,4,5)P(3)-binding pleckstrin homology domain is fully functional at inducing cell elongation and migration, suggesting that the main function of DHR-1 is to bind PtdIns(3,4,5)P(3). These results demonstrate that DOCK180, via its DHR-1 and DHR-2 domains, couples PtdIns(3,4,5)P(3) signalling to Rac GTP-loading, which is essential for directional cell movement.

p130(cas) (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival, and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs, including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by NMR spectroscopy, demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif. The serine-rich region bears strong structural similarity to four-helix bundles found in other adhesion components like focal adhesion kinase, alpha-catenin, or vinculin. Potential sites for phosphorylation and interaction with the 14-3-3 family of cellular regulators are identified in the domain and characterized by site-directed mutagenesis and binding assays. Mapping the degree of amino acid conservation onto the molecular surface reveals a patch of invariant residues near the C terminus of the bundle, which may represent a previously unidentified site for protein interaction.

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.

Cas-family proteins serve as docking proteins in integrin-mediated signal transduction. The founding member of this family, p130Cas, becomes tyrosine-phosphorylated in response to extracellular stimuli such as integrin-mediated cell adhesion and ligand engagement of receptor tyrosine kinases. Cas proteins are large multidomain molecules that transmit signals as intermediaries through interactions with signaling molecules such as FAK and other tyrosine kinases, as well as tyrosine phosphatases. After Cas is tyrosine-phosphorylated, it acts as a docking protein for binding SH2 domains of Src-family kinases. In order to examine the structural basis for a key step in propagation of signals by Cas, one of the major SH2-binding sites of Cas has been crystallized in complex with the SH3-SH2 regulatory domains of the Src-family kinase Lck. Crystallization conditions were identified by high-throughput screening and optimized with multiple rounds of seeding. The crystals formed at 295 K in space group P2(1)2(1)2(1), with unit-cell parameters a = 77.4, b = 107.3, c = 166.4 A, and diffract to 2.7 A resolution.