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 epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-␣ (TGF␣) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGF␣-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-␥1 (PLC-␥1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGF␣ stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) hydrolysis over those stimulated by TGF␣. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP 2 hydrolysis, PLC-␥1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-␥1 phosphorylation and PIP 2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-␥1 to its substrate in endocytic trafficking organelles.Cell signaling events mediated by epidermal growth factor receptor (EGFR) 1 regulates survival, proliferation, migration, and differentiation of many cell types. At least five ligands are known to activate EGFR, including epidermal growth factor (EGF) and transforming growth factor ␣ (TGF␣). Progress has been made in the last two decades in elucidating structurefunction relationships for EGFR and other receptor tyrosine kinases, particularly in how signal transduction is modulated by self-phosphorylation of cytoplasmic tyrosine residues (1). This permits access to the kinase domain of EGFR (2) and allows the receptor to bind signaling proteins containing modular Src homology 2 (SH2) and phosphotyrosine-binding domains (3, 4). Such interactions can affect the activity of the bound protein through transmission of conformational changes, enhancement of tyrosine phosphorylation, and/or localization in proximity to membrane-associated target molecules. One of the prominent signaling proteins activated by EGFR is the ␥1 isoform of phospholipase C (PLC) (5). This enzyme, which has two SH2 domains, catalyzes the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ), generating the second messengers diacylglycerol and inositol triphosphate and liberating PIP 2 -bound proteins (6). PLC-␥1 activity is positively modulated ...

To elucidate the complex structure of the human immunoglobulin lambda gene locus, a 1020-kb contig was constructed using 184 cosmid clones and one bacterial artificial chromosome (BAC) clone. A high-resolution physical map of this contig revealed that the entire lambda gene locus is 911 kb in length. It contains seven constant region (C lambda) gene segments and 69 unique EcoRI-HindIII segments that hybridize to variable region gene (V lambda) probes. The VpreB gene, BCRL4, and gamma-glutamyl transpeptidase gene (GGT)-like sequences are also located within the lambda gene locus. Hybridization analysis suggested that the lambda gene locus has undergone extensive amplification events in evolution.

The majority of pediatric anaplastic large cell lymphomas (ALCLs) carry the t(2;5)(p23;q35) chromosomal translocation that juxtaposes the dimerization domain of nucleophosmin with anaplastic lymphoma kinase (ALK). The nucleophosmin-ALK fusion induces constitutive, ligand-independent activation of the ALK tyrosine kinase leading to aberrant activation of cellular signaling pathways. To study the early consequences of ectopic ALK activation, a GyrB-ALK fusion was constructed that allowed regulated dimerization with the addition of coumermycin. Lymphoma is the third most common cancer in children and adolescents in the United States, representing 13% of newly diagnosed malignancies in the pediatric age group.1 Although significant progress has been made in the treatment and outcome of childhood lymphoma, many children still succumb to disease, or suffer acute and long-term toxicity from contemporary multiagent chemotherapy.2-4 A subtype of pediatric anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive lymphoma (ALKϩ lymphoma), may serve as an ideal model for the study of the molecular mechanisms of malignant transformation.5-7 ALKϩ lymphoma is characterized by a t(2;5)(p23;q35) chromosomal translocation that creates a chimeric fusion protein consisting of the amino-terminal portion of the nucleolar phosphoprotein, nucleophosmin (NPM), and the cytoplasmic domain of the receptor tyrosine kinase, ALK. 8,9 ALK is a recently described receptor tyrosine kinase in the insulin receptor subfamily whose expression is normally restricted to the central nervous system. 10 -12 NPM is a ubiquitously expressed homohexameric nucleolar phosphoprotein that shuttles ribosomal proteins between the nucleus and cytoplasm. [13][14][15] Essential functions of NPM in the chimera are provision of both a promoter that drives ectopic expression of ALK in lymphoid cells, and a dimerization motif that facilitates trans-phosphorylation and hence activation of signaling. 16The t(2;5) translocation is an essential event in the pathogenesis of ALKϩ lymphoma as NPM-ALK potently transforms both rat fibroblast and murine lymphoid cell lines, and induces lymphoid tumors in mice.17,18 NPM-ALK homodimers autophosphorylate on multiple residues, creating docking sites for several SH2 domaincontaining signaling molecules. 16,19 -21 Formation of this signaling complex leads to changes in gene expression that underlie the malignant phenotype. One goal of our studies is to understand the changes in cellular transcription that the NPM-ALK fusion protein produces.Ligand-induced dimerization is thought to directly induce the activation of receptor tyrosine kinases. [22][23][24] Numerous researchers have generated protein fusions that can be conditionally dimerized in the presence of bivalent compounds such as coumermycin, rapamycin, and FK1012. [25][26][27] To better define the immediate early effects of ALK activation, we used this technology to generate a conditionally dimerizable kinase construct, designed to mimic the activity of...

Transglutaminase 2 (TG2) is a ubiquitously expressed protein that catalyzes protein/protein crosslinking. Because extracellular TG2 crosslinks components of the extracellular matrix, TG2 is thought to function as a suppressor of cellular invasion. We have recently uncovered that the TG2 gene (TGM2) is a target for epigenetic silencing in breast cancer, highlighting a molecular mechanism that drives reduced TG2 expression, and this aberrant molecular event may contribute to invasiveness in this tumor type. Because tumor invasiveness is a primary determinant of brain tumor aggressiveness, we sought to determine if TGM2 is targeted for epigenetic silencing in glioma. Analysis of TGM2 gene methylation in a panel of cultured human glioma cells indicated that the 5′ flanking region of the TGM2 gene is hypermethylated and that this feature is associated with reduced TG2 expression as judged by immunoblotting. Further, culturing glioma cells in the presence of the global DNA demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor Trichostatin A resulted in re-expression of TG2 in these lines. In primary brain tumors we observed that the TGM2 promoter is commonly hypermethylated and that this feature is a cancer-associated phenomenon. Using publically available databases, TG2 expression in gliomas was found to vary widely, with many tumors showing overexpression or underexpression of this gene. Since overexpression of TG2 leads to resistance to doxorubicin through the ectopic activation of NFκB, we sought to examine the effects of recombinant TG2 expression in glioma cells treated with commonly used brain tumor therapeutics. We observed that in addition to doxorubicin, TG2 expression drove resistance to CCNU; however, TG2 expression did not alter sensitivity to other drugs tested. Finally, a catalytically null mutant of TG2 was also able to support doxorubicin resistance in glioma cells indicating that transglutaminase activity is not necessary for the resistance phenotype.