Showing papers on "Cyclin-dependent kinase 8 published in 2001"

Nuclear localization of EGF receptor and its potential new role as a transcription factor

Abstract: Epidermal growth factor receptor (EGFR) has been detected in the nucleus in many tissues and cell lines. However, the potential functions of nuclear EGFR have largely been overlooked. Here we demonstrate that nuclear EGFR is strongly correlated with highly proliferating activities of tissues. When EGFR was fused to the GAL4 DNA-binding domain, we found that the carboxy terminus of EGFR contained a strong transactivation domain. Moreover, the receptor complex bound and activated AT-rich consensus-sequence-dependent transcription, including the consensus site in cyclin D1 promoter. By using chromatin immunoprecipitation assays, we further demonstrated that nuclear EGFR associated with promoter region of cyclin D1 in vivo. EGFR might therefore function as a transcription factor to activate genes required for highly proliferating activities.

Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking.

Abstract: Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

Abstract: The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC50s 75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Tyrosine kinase inhibitors-ZD1839 (Iressa).

Abstract: Several epithelial tumors display epidermal growth factor receptor (EGFR) overexpression (with or without EGFR gene amplification) that is often associated with increased production of EGFR ligands. This permits the activation of endogenous tumor EGFR via autocrine mechanisms, resulting in cellular proliferation and tumor growth. Interruption of receptor signaling with bivalent EGFR antibodies or with small molecule inhibitors of the EGFR tyrosine kinase results in inhibition of tumor cell proliferation or viability in vitro and in vivo. One small molecule currently undergoing preclinical and clinical investigation is ZD1839 (Iressa), a synthetic anilinoquinazoline capable of inhibiting EGFR tyrosine kinase in vitro. The early results of clinical trials indicate this drug possesses antitumor activity in certain malignancies of the upper aerodigestive tract.

Mig-6 is a negative regulator of the epidermal growth factor receptor signal.

Abstract: In contrast to signal generation and transmission, the mechanisms and molecules that negatively regulate receptor tyrosine kinase (RTK) signaling are poorly understood. Here we characterize Mig-6 as a novel negative feedback regulator of the epidermal growth factor receptor (EGFR) and potential tumor suppressor. Mig-6 was identified in a yeast two-hybrid screen with the kinase active domain of the EGFR as bait. Upon EGF stimulation Mig-6 binds to the EGFR involving a highly acidic region between amino acids 985-995. This interaction is kinase activity-dependent, but independent of tyrosine 992. Mig-6 overexpression results in reduced activation of the mitogenactivated protein kinase ERK2 in response to EGF, but not FGF or PDGF, stimulation and in enhanced receptor internalization without affecting the rate of degradation. The induction of Mig-6 mRNA expression in response to EGF, but not FGF, indicates the existence of a negative regulatory feedback loop. Consistent with these findings, a possible role as tumor suppressor is indicated by Mig-6-mediated inhibition of EGFR overexpression-induced transformation of Rati cells.

Suppressor and oncogenic roles of transforming growth factor-β and its signaling pathways in tumorigenesis

Abstract: Transforming growth factor-β (TGF-β) has been implicated in oncogenesis since the time of its discovery almost 20 years ago. The complex, multifunctional activities of TGF-β endow it with both tumor suppressor and tumor promoting activities, depending on the stage of carcinogenesis and the responsivity of the tumor cell. Dysregulation or alteration of TGF-β signaling in tumorigenesis can occur at many different levels, including activation of the ligand, mutation or transcriptional suppression of the receptors, or alteration of downstream signal transduction pathways resulting from mutation or changes in expression patterns of signaling intermediates or from changes in expression of other proteins which modulate signaling. New insights into signaling from the TGF-β receptors, including the identification of Smad signaling pathways and their interaction with mitogen-activated protein (MAP) kinase pathways, are providing an understanding of the changes involved in the change from tumor suppressor to tumor promoting activities of TGF-β. It is now appreciated that loss of sensitivity to inhibition of growth by TGF-β by most tumor cells is not synonymous with complete loss of TGF-β signaling but rather suggests that tumor cells gain advantage by selective inactivation of the tumor suppressor activities of TGF-β with retention of its tumor promoting activities, especially those dependent on cross talk with MAP kinase pathways and AP-1.

Notch ligand‐bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling

Abstract: Thymic epithelial cells are specialized to play essential roles at multiple stages of T cell development in the thymus, yet the molecular basis of this specialization is largely unknown. Recently, the Notch family of transmembrane proteins has been implicated in thymocyte development. Such proteins interact with cell surface proteins of the Delta-like and Jagged families. It is known that Notch ligands are expressed intrathymically, and that Notch signaling is regulated by Notch ligands expressed on either the same or third-party cells. However, functional analysis of Notch ligand expression, and elucidation of the mechanism of Notch ligand signaling in thymocyte development, are unclear. Here, we find that Notch ligand expression in the thymus is compartmentalized, with MHC class II(+) thymic epithelium, but not thymocytes nor dendritic cells, expressing Jagged-1, Jagged-2 and Delta-like-1. We also provide evidence that contact with Notch ligands on thymic epithelium is necessary to activate and sustain Notch signaling in thymocytes, and that this can occur independently of positive selection induction. Our data suggest that Notch ligand expression by thymic epithelium may partly explain the specialization of these cells in supporting thymocyte development, by regulating Notch activation via an inductive signaling mechanism independently of signaling leading to positive selection.

Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

Abstract: The Saccharomyces cerevisiae Mediator complex was originally identified because of its ability to stimulate activated transcription in vitro. Many of the subunits of the purified Mediator complex (24) are encoded by SRB genes, first characterized as suppressors of a deletion in the C-terminal heptapeptide repeat (CTD) of the large subunit of RNA polymerase II (Pol II) (23). Additional Mediator subunits are encoded by genes initially identified in other genetic screens for mutations affecting gene control and are named accordingly (e.g., RGR-1). Mediator subunits not characterized previously were called Med1, Med2, etc. (24). Mediator subunits in yeast have also been purified as part of a still larger holoenzyme complex including Pol II and several general transcription factors (GTFs) (23). The Pol II holoenzyme analyzed by Young and colleagues (see reference 23 for a review) includes Srb8, Srb9, Srb10, and Srb11, whereas the Mediator complex studied by Myers et al. (24) lacks these subunits. The Srb8 to Srb11 subunits form a functional subcomplex of the holoenzyme required for repression by several yeast repressors (3, 11, 23). These subunits are regulated differently from other yeast Mediator and holoenzyme subunits. The intracellular Srb10 concentration falls dramatically as yeast cells deplete nutrients from their media, whereas the concentrations of other Mediator subunits do not (11). Recently, Liu et al. analyzed yeast Mediator complexes in a nuclear extract, avoiding ion-exchange chromatography and high salt concentration to avoid dissociation of subunits (18). Under these conditions they found that the majority of each Mediator subunit, including Srb8 to Srb11, was associated with Pol II in a complex of ∼1.9 MDa that lacks GTFs. A less abundant complex of ∼0.55 MDa included a subset of Mediator subunits. Several mammalian multiprotein complexes have been identified that have several subunits homologous to components of the yeast Mediator and several subunits that are not clearly related to yeast proteins (21). Broadly speaking, two size classes of complexes have been identified. Complexes of ∼2 MDa, such as the TRAP/SMCC (12), NAT (32), DRIP (30), ARC (25), and human Mediator (2) complexes, share an overlapping set of components. Smaller complexes (∼500 to 700 kDa) containing Srb/Med homologs have also been identified, including the murine Mediator (13), CRSP (31), and PC2 (20) complexes. These mammalian Mediator-like complexes were identified and purified by different biochemical procedures. TRAP (7), DRIP (30), ARC (25), and human Mediator (2) were purified on the basis of their ability to bind to activation domains during affinity chromatography. SMCC (8) and NAT (32) were purified based on their content of CDK8, which is a homolog of yeast Srb10. Functions of these Mediator complexes were assayed in different in vitro transcription systems that varied in the purity of the GTFs and the use of naked DNA versus chromatin templates. Most of these complexes, including ARC, DRIP, PC2, and CRSP, greatly stimulated activated transcription (21). NAT and SMCC repressed activated transcription in assays with highly purified factors (8, 32), but SMCC activated transcription when TFIIH was omitted (8). This repression has been attributed to the phosphorylation of the cyclin H subunit of TFIIH by the CDK8 kinase within the Mediator complexes (1). The human Mediator complex inhibited activated transcription in a highly purified system but stimulated high levels of activated transcription in reactions with partially purified GTFs (2). While the different mammalian Mediator-like complexes so far described share many subunits, they also differ with regard to their reported subunit composition (21). This raises the question whether there are multiple distinct Mediator-like complexes in mammalian cells that may differ in their functional properties or whether there is in fact one or a small number of mammalian Mediator complexes. In the latter case, the apparent differences in subunit composition reported by different laboratories might result from relatively minor differences in the methods used to characterize the subunits or from different methods of purification that partially dissociate a single large complex. To estimate the number of different complexes containing Mediator subunits in HeLa cells, we subjected unfractionated HeLa cell nuclear extract to gel filtration chromatography at low salt concentration to avoid the dissociation of subunits. Protein complexes in eluted fractions were characterized by immunoblotting with several antibodies specific for Mediator subunits, including components of both size classes of Mediator complexes described. A single peak of ∼2 MDa containing each of the several Mediator subunits was observed. No significant peak was observed at ∼500 to 700 kDa, indicating that either this size class of Mediator complex is much less abundant than the ∼2-MDa size class or that the ∼500 to 700-kDa Mediator complexes described above were derived from the larger size class by dissociation during the multiple steps of column chromatography used in their purification. Mediator subunits CDK8, cyclin C, and hSur2 were also observed in lower-molecular-mass complexes, but only the ∼2-MDa size class significantly stimulated activated transcription. High-resolution gel filtration and immunoprecipitation analyses indicated that there are at least two subclasses of ∼2-MDa Mediator complexes, one containing CDK8 and cyclin C and one lacking these subunits. A total of ∼300,000 hSur2 subunits per cell were present in the ∼2-MDa Mediator complexes; this number is approximately equal to the number of Pol II molecules per HeLa cell (15).

p53 Tumor Suppressor Protein

Abstract: The p53 protein was originally identified during the late 1970s, by several independent groups, as a novel cellular protein that was tightly associated with the large T antigen in cells transformed by simian virus-40 (SV40) (1–3). Although originally thought to function as an oncogene, isolation of the wild-type (wt) gene encoding p53 led to the discovery that p53 functioned as a potent tumor suppressor (4–8). A role for p53 in preventing malignant progression was subsequently demonstrated by the observations that transfection of p53 into cultured cells inhibited transformation by a number of oncogenes (9), and that mice lacking the p53 gene rapidly developed tumors with high incidence (10–12). It is now known that p53 is one of the most frequently mutated genes in human cancer, in which loss of function mutations contribute to the development of many major human malignancies. Approximately 50% of all human tumors carry a p53 mutation, and at least 52 different types of tumor have p53 mutations (13–15). During the past decade, p53 has been brought to the forefront of cancer research, and intensive investigation has provided insight into how it mediates its tumor suppressor activities, and how these activities are regulated. Elucidation of the mechanisms that activate and regulate p53, and the identification of upstream and downstream effectors and targets involved in p53 function, should contribute to understanding how cancers arise, and to the development of new therapeutic tools for their treatment.

EGFR as a transcription factor

Abstract: This article has been retracted at the request of the editor. Reason: This article by R.W.C. Wong and S.Y. Chan entitled ‘EGFR as a transcription factor?', has been retracted because it contains large tracts of text that have been copied from a previously published Nature Cell Biology News and Views article by Mark Waugh and Justin Hsuan [Nat. Cell Biol. (2001) 3 E209–E211].

Regulation of Epidermal Growth Factor Receptor Signaling by cbl-b

Abstract: : Negative regulation of the Epidermal Growth Factor Receptor (EGFR) signaling is essential to proper regulation of cellular function. Genetic evidence from C. elegans and Drosophila melanogaster has demonstrated that cbl proteins are negative regulators of the EGFR in these organisms. To determine the role of mammalian cbl proteins in EGFR signaling. stable clones overexpressing cbl-b were created in two cell lines which have distinct biological responses to EGFR activation. In the 3.2DIEGFR murine hematopoetic cell line. overexpression of cbl-b inhibits Epidermal Growth Factor-induced (EGF) proliferation. In the MDA-MB-468 human breast cancer cell line. EGFR activation induces apoptosis. Overexpression of cbl-b in these cells inhibits EGF-induced apoptosis. These data demonstrate that the mammalian cbl-b protein. like the C. elegans and Drosophila homologs. inhibits EGFR function. The molecular basis of this inhibition was studied in these two model systems. cbl-b is phosphorylated and recruited to the EGFR upon activation. In both cell lines. activation of the EGFR and activation of multiple downstream pathways have a shortened duration of signaling when cbl-b is overexpressed. Further biochemical analysis demonstrated that cbl-b increased activation-induced downregulation of the EGFR by enhancing EGFR ubiquitination and EGFR degradation. Specific inhibitors of either lysosomal or proteasomal proteases blocked cbl-b mediated EGFR degradation. Further analysis of cbl-b expression after cells are stimulated with EGF demonstrated that cbl-b is coordinately degraded along with the EGFR. Both EGFR and cbl-b downregulation requires an intact Tyrosine Kinase Binding and RING finger domain of the cbl-b protein. Additionally. binding of cbl-b to the EGFR is required for either protein to undergo EGF-induced degradation. Other proteins which are recruited to the activated EGFR complex are also coordinately degraded with the EGFR and cbl-b.