Kermit L. Carraway

ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor.

Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (PtdIns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of PtdIns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated PtdIns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, PtdIns 3-kinase coprecipitates with the ErbB3 protein (p180erbB3) in response to EGF. p180erbB3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of PtdIns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.

Met Receptor Contributes to Trastuzumab Resistance of Her2-Overexpressing Breast Cancer Cells

Her2 is overexpressed in 20% to 30% of breast tumors and correlates with reduced disease-free and overall patient survival. Trastuzumab, a humanized monoclonal antibody directed against Her2, represents the first Her2-targeted therapy, which decreases the risk of relapse and prolongs patient survival. Resistance to trastuzumab, both inherent and treatment-acquired, represents a significant barrier to the effective treatment of Her2 (+) breast cancer. The Met receptor tyrosine kinase is aberrantly expressed in breast cancer and predicts poor patient prognosis. In this study, we find that Met is frequently expressed in Her2-overexpressing breast cancer cells, as well as Her2 (+) breast cancer. Importantly, Met contributes to trastuzumab resistance, as inhibition of Met sensitizes cells to trastuzumab-mediated growth inhibition, whereas Met activation protects cells against trastuzumab by abrogating p27 induction. Remarkably, Her2-overexpressing breast cancer cells rapidly up-regulate Met expression after trastuzumab treatment, promoting their own resistance. Our study suggests that a subset of Her2 (+) patients may benefit from combined inhibition of Her2 and Met.

The Epidermal Growth Factor Receptor Regulates Interaction of the Human DF3/MUC1 Carcinoma Antigen with c-Src and β-Catenin

The DF3/MUC1 mucin-like, transmembrane glycoprotein is aberrantly overexpressed in most human carcinomas. The MUC1 cytoplasmic domain interacts with the c-Src tyrosine kinase and thereby increases binding of MUC1 and β-catenin. In the present work, coimmunoprecipitation studies demonstrate that MUC1 associates constitutively with the epidermal growth factor receptor (EGF-R) in human ZR-75-1 breast carcinoma cells. Immunofluorescence studies show that EGF-R and MUC1 associate at the cell membrane. We also show that the activated EGF-R phosphorylates the MUC1 cytoplasmic tail on tyrosine at a YEKV motif that functions as a binding site for the c-Src SH2 domain. The results demonstrate that EGF-R-mediated phosphorylation of MUC1 induces binding of MUC1 to c-Src in cells. Moreover, in vitro and in vivo studies demonstrate that EGF-R increases binding of MUC1 and β-catenin. These findings support a novel role for EGF-R in regulating interactions of MUC1 with c-Src and β-catenin.

Cytokinins: Structure/activity relationships☆

Abstract Sixty-nine compounds, mostly purine derivatives and closely related substances, were tested for promotion of growth and regulation of organ formation in the tobacco bioassay to determine relationships between chemical structure and cytokinin activity. Forty-three substances were synthesized in this study, and 13 of these were reported for the first time. N 6 -Alkyladenines (I) varied in activity over a wide concentration range depending on the length of the alkyl chain. Starting with adenine, detectable at ⩾200 μM, activity increased with the chain length to an optimum for 6-pentylaminopurine detectable at ca. 0–001 μM, and then decreased to reach a barely detectable level for 6-decylaminopurine. The result of the incorporation of polar groups in the side chain was not necessarily reduction in activity. One hydroxyl group, as in zeatin (Id), improved the activity of 6-(γ,γ-dimethylallylamino)purine (Ib) if it affected it at all; two hydroxyl groups, as in 6-(2,3-dihydroxy-3-methylbutylamino)purine strongly reduced activity. Comparisons of 6-isoamylaminopurine with 6-(γ,γ-dimethylallylamino)purine and of other closely related pairs of compounds showed that a double bond in the side chain greatly increased cytokinin activity. Adenine derivatives with cyclic substituents in the N 6 -position (benzyl-Ic), cyclohexyl-, etc.) showed the same general range of activity, potentiation by unsaturation, and variation in activity with substituent size, etc. as did the alkyl derivatives. Heteroatoms in or on the substituent groups decreased activity (in the case of N or Cl) or had little effect (S for O in furfuryl). Of the mono-substituted adenines only the N 6 -derivatives definitely possessed cytokinin activity. The 1-(III), 3-(II), or 9-substituted adenines probably are inactive but could be activated by conversion to the N 6 -isomers. Except for slight activity in tests of high concentrations, which could be ascribed to contaminants, 7-substituted adenines were completely inactive. Modification in the adenine moiety lowered the cytokinin activity, often by 95 per cent or more. Substitution of N for the 8-C atom in kinetin and in 6-benzylaminopurine or S for the 6-amino N atom in 6-(γ,γ-dimethylallylamino)purine did not eliminate but drastically reduced activity in the tobacco bioassay. Elimination of the 6-amino group without substituting another group completely removed activity; thus, the purine derivatives, 1-benzylpurine and 1-(γ,γ-dimethylallyl)purine, were inactive in tests where the 1-adenine derivatives could be activated to give a positive response. Addition of a second substituent on the 1-or 3-position of N 6 -substituted adenines drastically reduced or eliminated cytokinin activity. It is suggested that the 1-position and possibly also the 3-position must be free. A second substituent in the N 6 -, 7-, or 9-position of N 6 -substituted adenine derivatives lowered but did not eliminate activity. Also, the disubstituted 1-adenine derivatives, 1,9-dibenzyladenine and 1,7-dibenzyladenine were active, presumably after rearrangement to the corresponding N 6 -substituted isomers.

THE TRANSMEMBRANE MOLECULE KEKKON 1 ACTS IN A FEEDBACK LOOP TO NEGATIVELY REGULATE THE ACTIVITY OF THE DROSOPHILA EGF RECEPTOR DURING OOGENESIS

We have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.

AN INTRAMEMBRANE MODULATOR OF THE ERBB2 RECEPTOR TYROSINE KINASE THAT POTENTIATES NEUREGULIN SIGNALING

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Neuregulins and their receptors.

The recent identification of an activator for the ErbB2/Neu receptor has uncovered a new family of polypeptide growth factors that undoubtedly play a major role in the regulation of neuronal growth and differentiation. These factors, called the neuregulins, are expressed in neural and mesenchymal tissues, and activate members of the epidermal growth factor family of receptor tyrosine kinases. The identification and characterization of the neuregulins and their receptors will facilitate the dissection of the biochemical pathways regulating nervous system development.

Rat Muc4 (sialomucin complex) reduces binding of anti‐ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance

Muc4 (also called sialomucin complex), the rat homolog of human MUC4, is a heterodimeric glycoprotein complex that consists of a peripheral O‐glycosylated mucin subunit, ASGP‐1, tightly but noncovalently linked to a N‐glycosylated transmembrane subunit, ASGP‐2. The complex is expressed in a number of normal, vulnerable epithelial tissues, including mammary gland, uterus, colon, cornea and trachea. Muc4/SMC is also overexpressed or aberrantly expressed on a number of human tumors including breast tumors. Overexpression of Muc4/SMC has been shown to block cell‐cell and cell‐matrix interactions, protect tumor cells from immune surveillance and promote metastasis. In addition, as a ligand for ErbB2, Muc4/SMC can potentiate phosphorylation of ErbB2 and potentially alter signals generated from this receptor. Using A375 human melanoma cells and MCF7 human breast adenocarcinoma cells stably transfected with tetracycline regulatable Muc4, we have investigated whether overexpression of Muc4/SMC can repress antibody binding to cell surface‐expressed ErbB2. Overexpression of Muc4/SMC does not affect the level of ErbB2 expression in either cell line, but it does reduce binding of a number of anti‐ErbB2 antibodies, including Herceptin. Interestingly, overexpression of ErbB2 does not block binding of other unrelated antibodies of the same isotype, suggesting that the reduction in ErbB2 antibody binding is due to complex formation of Muc4/SMC and ErbB2. Furthermore, capping of Muc4/SMC with anti‐Muc4/SMC antibodies reduces antibody binding to ErbB2 instead of increasing binding, again suggesting that reduced antibody binding to ErbB2 is due to steric hindrance from complex formation of Muc4/SMC and ErbB2. Thus, overexpression of Muc4/SMC on tumor cells may have both prognostic and therapeutic relevance.

Reversible Disruption of Cell-Matrix and Cell-Cell Interactions by Overexpression of Sialomucin Complex

Sialomucin complex (SMC) is a large, heterodimeric glycoprotein complex composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits and expressed abundantly on the cell surface of ascites 13762 rat mammary adenocarcinoma cells. We have isolated recombinant cDNAs containing different numbers of ASGP-1 mucin repeats, which can be expressed as protein products with variable lengths. To study the anti-adhesive effect of SMC, these cDNAs were transfected into human cancer cell lines. Using a tetracycline-responsive, inducible expression system, we demonstrated that the overexpression of SMC induces morphology changes, cell detachment, and cell-cell dissociation of transfected A375 human melanoma cells in culture. The transition between the adherent and suspension states of the cells is fully reversible and dependent on the SMC expression level. The anti-adhesion effect of SMC was further analyzed kinetically by measuring the cell adhesion of transfected A375 melanoma and MCF-7 breast cancer cell lines to fibronectin, laminin, and collagen IV, demonstrating that SMC disrupts integrin-mediated cell adhesion to extracellular matrix proteins. The degree of this anti-adhesion effect was dependent on the number of mucin repeats in the SMC molecule as well as the level of cell surface expression.

Stabilization of the E3 Ubiquitin Ligase Nrdp1 by the Deubiquitinating Enzyme USP8

ABSTRACT Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.