Muc4 serves as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The time to complex formation and the stoichiometry of the complex were determined to be <15 min and 1:1 by analyses of Muc4 and ErbB2 coexpressed in insect cells and A375 tumor cells. In polarized CACO-2 cells, Muc4 expression causes relocalization of ErbB2, but not its heterodimerization partner ErbB3, to the apical cell surface, effectively segregating the two receptors. The apically located ErbB2 is phosphorylated on tyrosines 1139 and 1248. The phosphorylated ErbB2 in CACO-2 cells recruits the cytoplasmic adaptor protein Grb2, consistent with previous studies showing phosphotyrosine 1139 to be a Grb2 binding site. To address the issue of downstream signaling from apical ErbB2, we analyzed the three MAPK pathways of mammalian cells, Erk, p38, and JNK. Consistent with the more differentiated phenotype of the CACO-2 cells, p38 phosphorylation was robustly increased by Muc4 expression, with a consequent activation of Akt. In contrast, Erk and JNK phosphorylation was not changed. The ability of Muc4 to segregate ErbB2 and other ErbB receptors and to alter downstream signaling cascades in polarized epithelial cells suggests that it has a role in regulating ErbB2 in differentiated epithelia. INTRODUCTIONErbB2 is a 185-kDa class I receptor tyrosine kinase that is structurally related to the epidermal growth factor receptor EGFR. The ErbB family of receptors includes four members: epidermal growth factor receptor (EGFR, HER1, or c-ErbB1), c-ErbB2 (HER2, p185 neu ), c-ErbB3 (HER3), and c-ErbB4 (HER4) (Riese and Stern, 1998), which share 40 -45% sequence identity (Stein and Staros, 2000). The ErbB receptor extracellular domains are composed of four subdomains, which in order from the N-terminus are known as I (L1), II (CR1), III (L2), and IV (CR2) (Bajaj et al., 1987;Lax et al., 1988;Ward et al., 1995); subdomains I and III and subdomains II and IV are homologous. ErbB ligand binding is mediated primarily by subdomain III, and to a lesser extent by subdomain I (Lax et al., 1989;Khoda et al., 1993). Subdomains II and IV mediate the formation of specific ErbB homo and heterodimers (Garrett et al., 2002;Ogiso et al., 2002). The extracellular domain is followed by a transmembrane domain, a tyrosine kinase domain, and a segment of variable length of ϳ200 amino acids containing several tyrosine phosphorylation sites. On activation, these phosphotyrosines become docking sites for cytoplasmic signaling proteins that, in turn, initiate characteristic downstream signaling events (Klapper et al., 2000). The pathways activated may lead the cell to proliferation, differentiation, or apoptosis (Alroy and Yarden, 1997;Riese and Stern, 1998). All ErbB family members, with the exception of ErbB2, have high-affinity soluble ligands that induce receptor homo-or heterodimer formation and phosphorylation and trigger downstream signaling. The preferred ErbB heterodimer partner is ErbB2, and the ErbB2-ErbB3 pair generates the strongest proliferative signal, even...
The membrane mucin Muc4 has been shown to alter cellular behavior through both anti-adhesive effects on cell-cell and cellextracellular matrix interactions and its ability to act as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The ERK pathway is regulated by both cell-matrix and cell-cell adhesion. An analysis of the effects of Muc4 expression on ERK phosphorylation in mammary tumor and epithelial cells, which exhibit both adhesion-dependent growth and contact inhibition of growth, showed that the effects are density dependent, with opposing effects on proliferating cells and contact-inhibited cells. In these cells, cell-matrix interactions through integrins are required for activation of the ERK mitogenesis pathway. However, cell-cell interactions via cadherins inhibit the ERK pathway. Expression of Muc4 reverses both of these effects. In contact-inhibited cells, Muc4 appears to activate the ERK pathway at the level of Raf-1; this activation does not depend on Ras activation. The increase in ERK activity correlates with an increase in cyclin D 1 expression in these cells. This abrogation of contact inhibition is dependent on the number of mucin repeats in the mucin subunit of Muc4, indicative of an anti-adhesive effect. The mechanism by which Muc4 disrupts contact inhibition involves a Muc4-induced relocalization of E-cadherin from adherens junctions at the lateral membrane of the cells to the apical membrane. Muc4-induced abrogation of contact inhibition may be an important mechanism by which tumors progress from an early, more benign state to invasiveness.
Abstract. Soil aggregate stability is a useful indicator of soil physical health and can be used to monitor condition through time. A novel method to quantify soil aggregate stability, based on the relative increase in the footprint area of aggregates as they disintegrate when immersed in water, has been developed and can be performed using a smartphone application – SLAKES. In this study the SLAKES application was used to obtain slaking index (SI) values of topsoil samples (0 to 10 cm) at 158 sites to assess aggregate stability in a mixed agricultural landscape. A large range in SI values of 0 to 7.3 was observed. Soil properties and land use were found to be correlated with observed SI values. Soils with clay content > 25 % and CEC : clay ratio > 0.5 had the highest observed SI values. Variation in SI for these soils was driven by OC content which fit a segmented exponential decay function. An OC threshold of 1.1 % was observed below which the most extreme SI values were observed. Soils under dryland and irrigated cropping had lower OC content and higher observed SI values compared to soils under perennial cover. These results suggest that farm managers can mitigate the effects of extreme slaking by implementing management practices to increase OC content, such as minimum tillage or cover-cropping. A regression-kriging method utilising a Cubist model with a suite of spatial covariates was used to map SI across the study area. Accurate predictions were produced with leave-one-out cross-validation (LOOCV) giving an LCCC of 0.85 and an RMSE of 1.1. Similar validation metrics were observed in an independent test set of samples consisting of 50 observations (LCCC = 0.82; RMSE = 1.1). The potential impact of implementing management practices that promote soil OC sequestration on SI values in the study area was explored by simulating how a 1 % increase in OC would impact SI values at observation points, and then mapping this across the study area. Overall, the maps produced in this study have the potential to guide management decisions by identifying areas that currently experience extreme slaking, and those areas that are expected to have a significant reduction in slaking by increasing OC content.
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