Cas Overexpression Research Articles

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development.

Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both β-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, leaves had sufficient rhodanese activity to compete with cyanide assimilation into amino acids. Using transgenic low cyanogen plants, it was shown that reducing root cyanogen levels is associated with elevated root nitrate reductase activity, presumably to compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted in up to a 50% increase in root total amino acids and a 9% increase in root protein accumulation. However, plant growth and morphology was altered in plants overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth.

The Cellular Apoptosis Susceptibility Protein (CAS) Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis and Cell Proliferation

A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization, leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1 following binding of cytochrome c. We report here that CAS expression itself is up-regulated during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces up-regulation of CAS in a posttranscriptional, caspase-8-dependent manner through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. We identified a novel signaling pathway whereby caspase-8 engages a feedforward cascade that leads to CAS up-regulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is up-regulated at both the mRNA and DNA levels in human breast tumors, consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS up-regulation in non-transformed cells. Intriguingly, oncogene-induced CAS up-regulation also resulted in greater susceptibility to TRAIL-induced cell death, consistent with its proapoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis and that overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

CRISPR transcript processing: a mechanism for generating a large number of small interfering RNAs.

BackgroundCRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) is a recently discovered prokaryotic defense system against foreign DNA, including viruses and plasmids. CRISPR cassette is transcribed as a continuous transcript (pre-crRNA), which is processed by Cas proteins into small RNA molecules (crRNAs) that are responsible for defense against invading viruses. Experiments in E. coli report that overexpression of cas genes generates a large number of crRNAs, from only few pre-crRNAs.ResultsWe here develop a minimal model of CRISPR processing, which we parameterize based on available experimental data. From the model, we show that the system can generate a large amount of crRNAs, based on only a small decrease in the amount of pre-crRNAs. The relationship between the decrease of pre-crRNAs and the increase of crRNAs corresponds to strong linear amplification. Interestingly, this strong amplification crucially depends on fast non-specific degradation of pre-crRNA by an unidentified nuclease. We show that overexpression of cas genes above a certain level does not result in further increase of crRNA, but that this saturation can be relieved if the rate of CRISPR transcription is increased. We furthermore show that a small increase of CRISPR transcription rate can substantially decrease the extent of cas gene activation necessary to achieve a desired amount of crRNA.ConclusionsThe simple mathematical model developed here is able to explain existing experimental observations on CRISPR transcript processing in Escherichia coli. The model shows that a competition between specific pre-crRNA processing and non-specific degradation determines the steady-state levels of crRNA and is responsible for strong linear amplification of crRNAs when cas genes are overexpressed. The model further shows how disappearance of only a few pre-crRNA molecules normally present in the cell can lead to a large (two orders of magnitude) increase of crRNAs upon cas overexpression. A crucial ingredient of this large increase is fast non-specific degradation by an unspecified nuclease, which suggests that a yet unidentified nuclease(s) is a major control element of CRISPR response. Transcriptional regulation may be another important control mechanism, as it can either increase the amount of generated pre-crRNA, or alter the level of cas gene activity.ReviewersThis article was reviewed by Mikhail Gelfand, Eugene Koonin and L Aravind.

Abstract 3177: Cas overexpression in breast cancer confers resistance to chemotherapy by evading apoptosis and overcoming G0/G1 arrest

Abstract Breast cancer patients treated with chemotherapy often develop resistance, resulting in highly aggressive tumors that are insensitive to drugs. Elucidating the mechanisms by which cancer cells are able to escape drug-induced apoptosis is thus paramount for developing better approaches to treat and cure breast cancer. Adriamycin is an anthracycline that is regularly used for treatment of breast cancer. Upon exposure to adriamycin, sensitive cells undergo cell death. However, as is the case for many chemotherapeutic drugs, cancer cells frequently develop resistance to adriamycin. We have previously reported that MCF-7 breast cancer cells that overexpress the adaptor molecule p130Cas (Cas) are resistant to adriamycin, whereas MCF-7 cells with endogenous levels of Cas are sensitive to the drug (Ta et al., 2006 Cancer Research). This is consistent with reports showing that high expression of Cas correlates with poor relapse-free and overall survival (Van der Flier et al., 2000 JNCI). Our group has shown that Cas overexpression results in significantly less apoptosis in the presence of adriamycin, and that, the kinase activities of c-Src and PI3K are required for this protection from apoptosis. Furthermore, we show that there is a significant increase in AKT activation when Cas-overexpressing cells are treated with adriamycin. Interestingly, while MCF-7 cells expressing endogenous levels of Cas exhibit a G1 arrest upon adriamycin treatment, the more resistant Cas-overexpressing cells effectively transit through G1 and accumulate in S-phase. This suggests that overexpression of Cas may allow damaged cells to bypass the G0/G1 DNA damage checkpoint. Indeed, both MCF-7 cells expressing either endogenous or elevated levels of Cas show similar degrees of DNA damage when treated with adriamycin, as measured by phospho-histone H2AX. We saw a similar progression through the G0/G1 checkpoint in Cas-overexpressing MCF-7 cells under conditions of serum deprivation, suggesting that high Cas expression may have a relatively global effect on G0/G1 cell cycle checkpoints in response to distinct cellular insults. Based on these data, we hypothesize that Cas overexpression enables breast cancer cells to proceed through the G0/G1 cell cycle arrest induced by conditions of stress, such as DNA damage or nutrient deprivation. Future studies will focus on determining mechanisms by which Cas overexpression allows cancer cells to bypass these cell cycle checkpoints. By gaining a better understanding of how cancer cells are able to adapt and continue to proliferate in an unfavorable environment, and develop resistance to chemotherapy, this study will further the development of better strategies for targeting this aggressive cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3177.

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130(Cas) (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.

A Novel Association between p130Cas and Resistance to the Chemotherapeutic Drug Adriamycin in Human Breast Cancer Cells

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Extracellular Matrix Rigidity Promotes Invadopodia Activity

Invadopodia are actin-rich subcellular protrusions with associated proteases used by cancer cells to degrade extracellular matrix (ECM) [1]. Molecular components of invadopodia include branched actin-assembly proteins, membrane trafficking proteins, signaling proteins, and transmembrane proteinases [1]. Similar structures exist in nontransformed cells, such as osteoclasts and dendritic cells, but are generally called podosomes and are thought to be more involved in cell-matrix adhesion than invadopodia [2-4]. Despite intimate contact with their ECM substrates, it is unknown whether physical or chemical ECM signals regulate invadopodia function. Here, we report that ECM rigidity directly increases both the number and activity of invadopodia. Transduction of ECM-rigidity signals depends on the cellular contractile apparatus [5-7], given that inhibition of nonmuscle myosin II, myosin light chain kinase, and Rho kinase all abrogate invadopodia-associated ECM degradation. Whereas myosin IIA, IIB, and phosphorylated myosin light chain do not localize to invadopodia puncta, active phosphorylated forms of the mechanosensing proteins p130Cas (Cas) and focal adhesion kinase (FAK) are present in actively degrading invadopodia, and the levels of phospho-Cas and phospho-FAK in invadopodia are sensitive to myosin inhibitors. Overexpression of Cas or FAK further enhances invadopodia activity in cells plated on rigid polyacrylamide substrates. Thus, in invasive cells, ECM-rigidity signals lead to increased matrix-degrading activity at invadopodia, via a myosin II-FAK/Cas pathway. These data suggest a potential mechanism, via invadopodia, for the reported correlation of tissue density with cancer aggressiveness.

CSE1L/CAS, a microtubule-associated protein, inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs

CSE1L/CAS, a microtubule-associated, cellular apoptosis susceptibility protein, is highly expressed in various cancers. Microtubules are the target of paclitaxel-induced apoptosis. We studied the effects of increased or reduced CAS expression on cancer cell apoptosis induced by chemotherapeutic drugs including paclitaxel. Our results showed that CAS overexpression enhanced apoptosis induced by doxorubicin, 5-fluorouracil, cisplatin, and tamoxifen, but inhibited paclitaxel-induced apoptosis of cancer cells. Reductions in CAS produced opposite results. CAS overexpression enhanced p53 accumulation induced by doxorubicin, 5-fluorouracil, cisplatin, tamoxifen, and etoposide. CAS was associated with alpha-tubulin and beta-tubulin and enhanced the association between alpha-tubulin and beta-tubulin. Paclitaxel can induce G2/M phase cell cycle arrest and microtubule aster formation during apoptosis induction, but CAS overexpression reduced paclitaxel-induced G2/M phase cell cycle arrest and microtubule aster formation. Our results indicate that CAS may play an important role in regulating the cytotoxicities of chemotherapeutic drugs used in cancer chemotherapy against cancer cells.

CAS Enhances Chemotherapeutic Drug-Induced p53 Accumulation and Apoptosis: Use of CAS for High-Sensitivity Anticancer Drug Screening

ABSTRACTCells attacked by cytotoxic toxins may express apoptosis-related proteins such as p53 to kill themselves, so as not to affect surrounding healthy cells. These apoptosis-related proteins are also crucial for inducing apoptosis of tumor cells in cancer chemotherapy. CSE1L/CAS is a cellular apoptosis susceptibility protein that plays important roles in mediating cell apoptosis induced by various cytotoxic toxins and chemotherapeutic drugs. Our studies showed that CAS over-expression increased p53 accumulation and apoptosis induced by 5-fluorouracil, doxorubicin, cisplatin, and tamoxifen in HT-29 cancer cells. A method based on coexpression of CAS with green fluorescence protein (GFP) was developed for high-sensitivity anticancer drug screening. Cancer cells transfected with CAS- and GFP-expressing vectors or the control and GFP-expressing vectors were grown on 96-well microplates, treated with compounds to be screened, and detected with a microplate fluorescence reader. GFP fluorescence decreased following cancer cell death induced by the anticancer compounds. CAS transfection enhanced the cytotoxicities of anticancer compounds and therefore increased the decline in GFP fluorescence. Thus, anticancer compounds could be identified more sensitively. Our study indicates that CAS is an important p53 and apoptosis regulator and may be used for high-throughput anticancer drug screening as well as cytotoxic toxin assays.

Evidence for chloroplast control of external Ca2+‐induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.

Physical and Functional Interactions between Cas and c-Src Induce Tamoxifen Resistance of Breast Cancer Cells through Pathways Involving Epidermal Growth Factor Receptor and Signal Transducer and Activator of Transcription 5b

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell–cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with β-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/β-catenin cell–cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.

IRF-1-Mediated CAS Expression Enhances Interferon-γ-Induced Apoptosis of HT-29 Colon Adenocarcinoma Cells

The expression of CAS is reported to be upregulated in a variety of human tumor cells, and such expression correlates with the development of tumors. CAS also plays a role in apoptosis. We investigated whether CAS expression affects the susceptibility of tumor cells to IFN-γ-induced apoptosis. Our data show that IFN-γ treatment induces CAS expression in HT-29 tumor cells. IFN-γ-induced gene expression is primarily mediated by the transcriptional factor, IRF-1. Our data show that IRF-1 mediates IFN-γ-induced CAS expression. Transfection of HT-29 cells with CAS expression vector did not induce apoptosis of cells; nevertheless, CAS overexpression greatly enhanced IFN-γ-induced apoptosis of cells. CPP32 is regarded as one of the central apoptosis executioner molecules. CAS overexpression enhances IFN-γ-induced CPP32 expression. These results indicate that tumor cells highly expressing CAS may be more susceptible to apoptosis induced by reagents that are capable of inducing CAS expression. Thus, CAS may be a target for the elimination of tumors.

Unraveling Cell Migration and Survival

Cell migration occurs under normal conditions and conditions of cellular dysregulation. Although it is known that the intracellular signals that lead to migration also suppress apoptosis, the molecular components required for both functions are not well characterized. Through the use of three-dimensional collagen matrix cell culture, Cho and Klemke identify several signaling proteins involved in migration and inhibition of apoptosis. COS-7 cells treated with growth factors migrated more and were spared from apoptosis, whereas untreated cells did not migrate and became apoptotic. Anti-integrin antibody treatment of COS-7 cells indicated an essential role for the integrin receptor in cellular migration and apoptosis inhibition. Overexpression of activated MEK, or the Crk-associated substrate (Cas) and Crk together in COS-7 cells greatly increased migration and survival. Similarly, overexpression of Cas and Crk mutants, which cannot associate, greatly diminished the cellular capacity to migrate and survive. The inactivation of ERK by the addition of a MEK inhibitor, in the presence of insulin or EGF but not fetal bovine serum (FBS), resulted in decreased migration and survival, indicating that FBS has components that promote cell migration and survival in an Erk-independent manner. Last, the authors identified that Rac activity is required for Cas/Crk-dependent migration and survival, whereas myosin light chain kinase (MLCK) is required for Erk-dependent functions. Thus, the activation of at least two independent pathways leads to migration and survival in cells. Cho, S.Y., and Klemke, R.L. (2000) Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. J. Cell Biol. 149 : 223-236. [Abstract] [Full Text]

Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling Pathways

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.

Cas Mediates Transcriptional Activation of the Serum Response Element by Src

The Src substrate p130(Cas) is a docking protein containing an SH3 domain, a substrate domain that contains multiple consensus SH2 binding sites, and a Src binding region. We have examined the possibility that Cas plays a role in the transcriptional activation of immediate early genes (IEGs) by v-Src. Transcriptional activation of IEGs by v-Src occurs through distinct transcriptional control elements such as the serum response element (SRE). An SRE transcriptional reporter was used to study the ability of Cas to mediate Src-induced SRE activation. Coexpression of v-Src and Cas led to a threefold increase in SRE-dependent transcription over the level induced by v-Src alone. Cas-dependent activation of the SRE was dependent on the kinase activity of v-Src and the Src binding region of Cas. Signaling to the SRE is promoted by a serine-rich region within Cas and inhibited by the Cas SH3 domain. Cas-dependent SRE activation was accompanied by an increase in the level of active Ras and in the activity of the mitogen-activated protein kinase (MAPK) Erk2; these changes were blocked by coexpression of dominant-negative mutants of the adapter protein Grb2. SRE activation was abrogated by coexpression of dominant-negative mutants of Ras, MAPK kinase (Mek1), and Grb2. Coexpression of Cas with v-Src enhanced the association of Grb2 with the adapter protein Shc and the protein tyrosine phosphatase Shp-2; coexpression of Shc or Shp-2 mutants significantly reduced SRE activation by Cas and v-Src. Cas-induced Grb2 association with Shp-2 and Shc may account for the Cas-dependent activation of the Ras/Mek/Erk pathway and SRE-dependent transcription. 14-3-3 proteins may also play a role in Cas-mediated signaling to the SRE. Overexpression of Cas was found to modestly enhance epidermal growth factor (EGF)-induced activation of the SRE. A Cas mutant lacking the Src binding region did not potentiate the EGF response, suggesting that Cas enhances EGF signaling by binding to endogenous cellular Src or another Src family member. These observations implicate Cas as a mediator of Src-induced transcriptional activation.

The Novel Catenin p120casBinds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts

p120cas(CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and β-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutantsin vivoindicated that the CAS–N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS–cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the stucturally similar β-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus β-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes.