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Results for “Multiprotein Complexes”

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PubMed2000

PU.1-mediated transcription is enhanced by HMG-I(Y)-dependent structural mechanisms.

The ets transcription factor PU.1 is an important regulator of the immunoglobulin heavy chain gene intronic enhancer, or mu enhancer. However, PU.1 is only one component of the large multiprotein complex required for B cell-specific enhancer activation. The transcriptional coactivator HMG-I(Y), a protein demonstrated to physically interact with PU.1, increases PU.1 affinity for the mu enhancer muB element, indicating that HMG-I(Y) may play a role in the transcriptionally active mu enhanceosome. Increased PU.1 affinity is not mediated by HMG-I(Y)-induced changes in DNA structure. Investigation of alternative mechanisms to explain the HMG-I(Y)-mediated increase in PU.1/mu enhancer binding demonstrated, by trypsin and chymotrypsin mapping, that interaction between PU.1 and HMG-I(Y) in solution induces a structural change in PU.1. In the presence of HMG-I(Y) and wild-type mu enhancer DNA, PU.1 becomes more chymotrypsin resistant, suggesting an additional change in PU.1 structure upon HMG-I(Y)-induced PU.1/DNA binding. From these results, we suggest that increased DNA affinity under limiting PU.1 concentrations is mediated by an HMG-I(Y)-induced structural change in PU.1. In functional assays, HMG-I(Y) further augments transcriptional synergy between PU.1 and another member of the ets family, Ets-1, indicating that HMG-I(Y) is a functional component of the active enhancer complex. These studies suggest a new mechanism for HMG-I(Y)-mediated coactivation; HMG-I(Y) forms protein-protein interactions with a transcription factor, which alters the three-dimensional structure of the factor, resulting in enhanced DNA binding and transcriptional activation. This mechanism may be important for transcriptional activation under conditions of limiting transcription factor concentration, such as at the low levels of PU.1 expressed in B cells.

3T3 Cells↗

PubMed2000

TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes.

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.

Adenosine Triphosphatases↗

PubMed2000

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Cloning and characterization of ELL-associated proteins EAP45 and EAP20. a role for yeast EAP-like proteins in regulation of gene expression by glucose.

RNA polymerase II elongation factor ELL was recently purified from rat liver as a component of a multiprotein complex containing ELL and three ELL-associated proteins (EAPs) of approximately 45 (EAP45), approximately 30 (EAP30), and approximately 20 (EAP20) kDa (Shilatifard, A. (1998) J. Biol. Chem. 273, 11212-11217). Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt, A. E., Miller, T., Schmidt, S. L., Shiekhattar, R., and Shilatifard, A. (1999) J. Biol. Chem. 274, 21981-21985), which is required for efficient derepression of glucose-repressed genes. Here we report the cloning of cDNAs encoding the EAP45 and EAP20 proteins. In addition, we identify the S. cerevisiae VPS36 and YJR102c genes as potential orthologs of EAP45 and EAP20 and show that they are previously uncharacterized SNF genes with properties very similar to SNF8.

Amino Acid Sequence↗

PubMed2001

TATA-binding protein-associated factors enhance the recruitment of RNA polymerase II by transcriptional activators.

Transcription factor (TF) IID, comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), is a general transcription factor required for RNA polymerase II (pol II) transcription on most eukaryotic genes. Recent findings that TAFs may not be globally required for activator-dependent transcription in vivo and in vitro and that both TAF-dependent and TAF-independent promoters are found in yeast suggest that transcriptional activation can occur through at least two different pathways, depending on the presence or absence of TAFs. Using order-of-addition and template challenge assays performed in a human cell-free transcription system reconstituted with recombinant general transcription factors (TFIIB, TBP, TFIIE, TFIIF), a recombinant general cofactor (PC4), and highly purified epitope-tagged multiprotein complexes (TFIID, TFIIH, pol II), we demonstrate that when TBP is used as the TATA-binding factor transcriptional activators such as Gal4-VP16 and human papillomavirus E2 mainly function by facilitating pol II entry to the promoter region. In contrast, when TFIID is used as the TATA-binding factor, promoter recognition by TFIID appears to be the rate-limiting step facilitated by transcriptional activators during preinitiation complex assembly. Using protein-protein pull-down and far-Western analyses, we further show that the presence of TAFs in TFIID facilitates the recruitment of pol II by transcriptional activators, thereby switching the rate-limiting step from pol II entry to promoter recognition. Our findings thus provide distinct molecular mechanisms for TAF-independent and TAF-dependent activation.

Animals↗

PubMed2001

A potential role for human cohesin in mitotic spindle aster assembly.

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

Animals↗

PubMed2001

Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme.

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

Biotin↗

PubMed2002

Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter.

Members of the Wnt family of secreted molecules have been established as key factors in determining cell fate and morphogenic signaling. It has long been recognized that Wnt induces morphogenic signaling through the Tcf/LEF-1 cascade by regulating free intracellular levels of beta-catenin, a co-factor for Tcf/LEF-1 transcription factors. In the present study, we have demonstrated that Wnt-3A can also directly induce transcription from the LEF-1 promoter. This induction was dependent on glycogen synthase kinase 3beta inactivation, a rise in free intracellular beta-catenin, and a short 110-bp Wnt-responsive element (WRE) in the LEF-1 promoter. Linear and internal deletion of this WRE led to a dramatic increase in constitutive LEF-1 promoter activity and loss of Wnt-3A responsiveness. In isolation, the 110-bp WRE conferred context-independent Wnt-3A or beta-catenin(S37A) responsiveness to a heterologous SV40 promoter. Studies expressing dominant active and negative forms of LEF-1, beta-catenin, GSK-3beta, and beta-catenin/LEF-1 fusions suggest that Wnt-3A activates the LEF-1 promoter through a beta-catenin-dependent and LEF-1-independent process. Wnt-3A expression also induced multiple changes in the binding of factors to the WRE and suggests that regulatory mechanisms may involve modulation of a multiprotein complex. In summary, these results provide evidence for transcriptional regulation of the LEF-1 promoter by Wnt and enhance the mechanistic understanding of Wnt/beta-catenin signaling in the regulation of LEF-1-dependent developmental processes.

Base Sequence↗

PubMed2002

Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback repressor pathway regulating Wnt signaling.

The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize beta-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3beta, and Axin1 or its homolog Axin2/Axil/conductin promotes beta-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant beta-catenin or gamma-catenin or after ligand-induced activation of a beta-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective beta-catenin regulation had elevated AXIN2 gene and protein expression. Expression of AXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of beta-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by beta-catenin-TCF. Because Axin2 can inhibit beta-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins.

Adenomatous Polyposis Coli Protein↗

PubMed2002

Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins.

The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Binding to Elongin C inhibits degradation of interacting proteins in yeast.

Elongin C is a highly conserved, low molecular weight protein found in a variety of multiprotein complexes in human, rat, fly, worm, and yeast cells. Among the best characterized of these complexes is a mammalian E3 ligase that targets proteins for ubiquitination and subsequent degradation by the 26 S proteasome. Despite its crucial role as a component of such E3 ligases and other complexes, the specific function of Elongin C is unknown. In yeast, Elongin C is a non-essential gene and there is no obvious phenotype as associated with its absence. We previously reported that in Saccharomyces cerevisiae Elongin C (Elc1) interacts specifically and strongly with a class of proteins loosely defined as stress response proteins. In the present study, we examined the role of yeast Elc1 in the turnover of two of these binding partners, Snf4 and Pcl6. Deletion of Elc1 resulted in decreased steady-state levels of Snf4 and Pcl6 as indicated by Western blot analysis. Northern blot analysis of mRNA prepared from elc1 null and wild type strains revealed no difference in mRNA levels for Snf4 and Pcl6 establishing that the effects of Elc1 are not transcriptionally mediated. Reintroduction of either yeast or human Elongin C into Elc1 null strains abrogated this effect. Taken together, these data document that the levels of Snf4 and Pcl6 are dependent on the presence of Elc1 and that binding to Elc1 inhibits the degradation of these proteins. The results suggest a new function for yeast Elongin C that is distinct from a direct role in targeting proteins for ubiquitination and subsequent proteolysis.

Animals↗

PubMed2002

SUMO-1 modification of histone deacetylase 1 (HDAC1) modulates its biological activities.

Histone deacetylation plays a central role in the regulation of genes linked to virtually all biological processes. This modification reaction is dependent on a family of related histone deacetylases (HDACs), which function as key components of large multiprotein complexes involved in the development of normal and neoplastic cells. The mechanisms regulating HDACs and their roles in such processes are not understood, and these form the major focus for the current study. Here, in the course of assessing possible post-translational modifications of HDAC1, we demonstrated that HDAC1 is a substrate for SUMO-1 (small ubiquitin-related modifier) modification in vitro and in vivo. The HDAC1 lysines targeted for modification were identified as C-terminal Lys-444 and Lys-476, which are also present in mammalian HDAC2 and lower vertebrate HDAC1/2 orthologs yet absent from other HDAC family members, pointing to a means of differential regulation among HDAC proteins. Mutation of these target residues (lysine to arginine substitution) profoundly reduced HDAC1-mediated transcriptional repression in reporter assays without affecting HDAC1 ability to associate with mSin3A and eliminated HDAC1-induced cell cycle and apoptotic responses upon overexpression. Together, the results demonstrate that HDAC1 is modified by SUMO-1, and this modification can dramatically affect HDAC1 activity in a number of surrogate biological assays.

3T3 Cells↗

PubMed2002

Evidence of functional modulation of the MEKK/JNK/cJun signaling cascade by the low density lipoprotein receptor-related protein (LRP).

Lipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-F(c) domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated alpha2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to alpha2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-kappaB) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun.

Animals↗

PubMed2002

Regulation of the human cyclin-dependent kinase inhibitor p18INK4c by the transcription factors E2F1 and Sp1.

The p18(INK4c) cyclin-dependent kinase inhibitor is an important regulator of cell cycle progression and cellular differentiation. We and others found that overexpressed E2F proteins up-regulate p18 expression. To better understand this phenomenon, we performed a functional analysis of the human p18 promoter. Deletion studies revealed that the E2F-responsive elements of the promoter are located within 131 bp upstream of the transcription start site. This region contains putative Sp1- and E2F-binding sites. Mutational inactivation of these elements revealed that the Sp1 sites were important for the basal activity of the promoter but could also mediate the effects of E2F1 on the p18 promoter. Moreover, we found that E2F1 and Sp1 can synergistically enhance the activity of the proximal p18 promoter. Gel shift analyses using p18 promoter-derived probes led to the identification of several multiprotein complexes that were found to contain different combinations of E2F proteins and/or Sp1. Recombinant E2F1 was also capable of binding to the E2F-binding sites. Chromatin immunoprecipitation experiments demonstrated that E2F1 and E2F4 associate with the p18 promoter in unperturbed cells. Based on these findings, we conclude that E2F proteins and Sp1 play an important role in the control of p18 expression.

Adenocarcinoma↗

PubMed2002

Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP.

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.

Amino Acid Sequence↗

PubMed2002

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells.

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G(2)-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34(cdc2) (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34(cdc2)-mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both gamma-tubulin and cytokeratin 19, p34(cdc2) phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase.

CDC2 Protein Kinase↗

PubMed2002

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97.

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Epidermal growth factor receptor-dependent and -independent cell-signaling pathways originating from the urokinase receptor.

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.

Animals↗