The RhoGAP protein Deleted in Liver Cancer 3 (DLC3) is essential for adherens junctions integrity.

Epithelial cell-cell contacts are mediated by E-cadherin interactions, which are regulated by the balanced local activity of Rho GTPases. Despite the known function of Rho at adherens junctions (AJs), little is known about the spatial control of Rho activity at these sites. Here we provide evidence that in breast epithelial cells the Deleted in Liver Cancer 3 (DLC3) protein localizes to AJs and is essential for E-cadherin function. DLC3 is a still poorly characterized RhoA-specific GTPase-activating protein that is frequently downregulated in various types of cancer. We demonstrate that DLC3 depletion leads to mislocalization of E-cadherin and catenins, which was associated with impaired cell aggregation and increased migration. This is explained by aberrant local Rho signaling because ROCK inhibition restored cell-cell contacts in DLC3 knockdown cells. We thus identify DLC3 as a novel negative regulator of junctional Rho and propose that DLC3 loss contributes to carcinogenesis by compromising epithelial integrity.

Update on HER-2 as a target for cancer therapy: intracellular signaling pathways of ErbB2/HER-2 and family members.

ErbB (also termed HER) receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin, in which they are involved in the control of diverse biological processes such as proliferation, differentiation, migration and apoptosis. Furthermore, their deregulated expression has been implicated in many types of human cancers and is associated with poor clinical prognosis. Owing to the importance of ErbB proteins in both development and cellular transformation, a lot of attention has been drawn to the intracellular signals initiated by the engagement of this family of receptor tyrosine kinases. This review will focus on the membrane proximal events triggered by the ErbB receptor network and will address questions of how receptor heterodimerization may contribute to signal specification and diversification.

The ErbB receptor tyrosine family as signal integrators.

ErbB receptor tyrosine kinases (RTKs) and their ligands have important roles in normal development and in human cancer. Among the ErbB receptors only ErbB2 has no direct ligand; however, ErbB2 acts as a co-receptor for the other family members, promoting high affinity ligand binding and enhancement of ligand-induced biological responses. These characteristics demonstrate the central role of ErbB2 in the receptor family, which likely explains why it is involved in the development of many human malignancies, including breast cancer. ErbB RTKs also function as signal integrators, cross-regulating different classes of membrane receptors including receptors of the cytokine family. Cross-regulation of ErbB RTKs and cytokines receptors represents another mechanism for controlling and enhancing tumor cell proliferation.

Characterization of an N-terminally truncated cyclin A isoform in mammalian cells.

Cyclin A is essential for regulating key transitions in the eukaryotic cell cycle including initiation of DNA replication and mitosis. This paper describes the characterization of a truncated cyclin A isoform (cyclin A(t)) in vitro in cultured mammalian cells and in mouse tissues. The presence of cyclin A(t) in specific cell types correlates with the ability of cell extracts to cleave in vitro translated cyclin A. In CHO-K1 cells, cyclin A processing to cyclin A(t) occurs at the N terminus; it does not involve the 26 S proteasome, nor could it be induced by conditional overexpression of the cyclin-dependent kinase inhibitor p27(Kip1). However, high cell densities lead to increased cyclin A(t) levels. Unlike full-length cyclin A, cyclin A(t) localizes to the cytoplasm, where it binds Cdk2. The data suggest that cyclin A processing occurs in vivo to yield an N-terminally truncated isoform by an unknown mechanism that is regulated by cell density. Differential subcellular localization may provide the first insights into the physiological role of cyclin A(t).

An essential role for Src kinase in ErbB receptor signaling through the MAPK pathway.

ErbB receptor tyrosine kinases are activated by multiple ligands such as epidermal growth factor (EGF) and neuregulins (NRGs), leading to stimulation of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. We show here that Src kinase is essential for rapid EGF- and NRG-induced MAPK activation when the breast carcinoma cell lines T47D and SKBR3 are stimulated with low concentrations of ligand. In the presence of the pharmacological inhibitor CGP77675, which specifically blocks the activity of Src family kinases, ligand-induced MAPK activation was almost completely blocked at 5 min. Although this block was only transient, inactivation of Src suppressed ligand-induced transcription from a MAPK-responsive promoter. At the molecular level, the initial inhibition of MAPK by Src inactivation correlated with impaired ligand-induced Shc phosphorylation. Surprisingly, Src inhibition affected neither association of Shc with ErbB receptors nor phosphorylation of receptor-bound Shc. Thus, ErbB signaling requires the engagement of a novel Src-dependent route to MAPK, to trigger its rapid activation and subsequent efficient stimulation of transcription.

ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases.

Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr694) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF- and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases.

NDF/heregulin-induced cell cycle changes and apoptosis in breast tumour cells: role of PI3 kinase and p38 MAP kinase pathways.

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2-3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.

ErbB-1 and ErbB-2 acquire distinct signaling properties dependent upon their dimerization partner.

The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.