ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells: involvement of PI3K and Erk signaling.

The neuroprotective effects of neuregulin (NRG), a polypeptide growth factor, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death and oxidative stress in PC12-ErbB4 cells were investigated. Treatment of PC12-ErbB4 cells with MPP+ induced cell death that was markedly attenuated by NRG. The PI3K/PKB/Akt and Ras/MapK signaling pathways probably mediate the survival effect of NRG. NRG induces prolonged activation of PKB/Akt and Erk. Moreover, inhibition of the PI3K and MEK activities prevented the NRG-induced survival effect. Overexpression of constitutively active PI3K or H-Ras (12V) inhibited MPP+-mediated cell death. In addition, MPP+- mediated reactive oxygen species (ROS) elevation was also inhibited by NRG. The effect of NRG on ROS levels was blocked by PI3K and MEK inhibitors, indicating that both signaling pathways can regulate the toxic ROS levels induced by MPP+. Taken together, these results indicate that in PC12-ErbB4 cells, the NRG-induced neuroprotective effect from MPP+ treatment, requires PI3K/PKB/Akt and Ras/MapK signaling networks.

Oncogenic synergism between ErbB1, nucleolin, and mutant Ras.

Alterations in the ErbB family of growth factor receptors, their signaling components, and mutational activation of Ras proteins are major contributors to malignant transformation. Recently, mutant Ras was shown to be capable of activating ErbB receptors in a ligand-independent manner. Furthermore, it was observed that nucleolin, a transcriptional regulator and ribosome biogenesis factor, can bind both K-Ras and the cytoplasmic tail of ErbB receptors to enhance ErbB receptor activation. However, the functional significance of these interactions to cancer pathogenesis has not been probed. Here, we show that endogenous nucleolin interacts simultaneously in vivo with endogenous Ras and ErbB1 (EGFR) in cancer cells. The C-terminal 212 amino acids of nucleolin were determined to be sufficient to interact with ErbB1 and all Ras protein isoforms (H-, N-, and K-Ras). Nucleolin partially colocalizes with Ras at the plasma membrane. Moreover, activated but not wild-type Ras facilitates nucleolin interaction with ErbB1 and stabilizes ErbB1 receptor levels. Most importantly, these three oncogenes synergistically facilitate anchorage-independent cell growth in vitro and tumor growth in vivo. Our findings suggest strategies to target nucleolin as a general approach to inhibiting ErbB- and Ras-driven cancers.

Structure-function analysis of nucleolin and ErbB receptors interactions.

BACKGROUND: The ErbB receptor tyrosine kinases and nucleolin are major contributors to malignant transformation. Recently we have found that cell-surface ErbB receptors interact with nucleolin via their cytoplasmic tail. Overexpression of ErbB1 and nucleolin leads to receptor phosphorylation, dimerization and anchorage independent growth. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we explored the regions of nucleolin and ErbB responsible for their interaction. Using mutational analyses, we addressed the structure-function relationship of the interaction between ErbB1 and nucleolin. We identified the ErbB1 nuclear localization domain as nucleolin interacting region. This region is important for nucleolin-associated receptor activation. Notably, though the tyrosine kinase domain is important for nucleolin-associated receptor activation, it is not involved in nucleolin/ErbB interactions. In addition, we demonstrated that the 212 c-terminal portion of nucleolin is imperative for the interaction with ErbB1 and ErbB4. This region of nucleolin is sufficient to induce ErbB1 dimerization, phosphorylation and growth in soft agar. CONCLUSIONS/SIGNIFICANCE: The oncogenic potential of ErbB depends on receptor levels and activation. Nucleolin affects ErbB dimerization and activation leading to enhanced cell growth. The C-terminal region of nucleolin and the ErbB1 NLS-domain mediate this interaction. Moreover, when the C-terminal 212 amino acids region of nucleolin is expressed with ErbB1, it can enhance anchorage independent cell growth. Taken together these results offer new insight into the role of ErbB1 and nucleolin interaction in malignant cells.

Identification of nucleolin as new ErbB receptors- interacting protein.

BACKGROUND: The ErbB receptor tyrosine kinases are major contributors to malignant transformation. These receptors are frequently overexpressed in a variety of human carcinomas. The role of the ErbB receptors and their ligands in carcinomas and the mechanism by which their overexpression leads to cancer development is still unclear. Ligand binding to specific ErbB receptor is followed by receptor dimerization, phosphorylation and recruitment of SH2 containing cytoplasmic proteins, which initiate the cascade of signaling events. Nevertheless, increasing data suggest that there are non-phosphorylated receptor-substrate interactions that may affect ErbB-mediated responses. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using GST-ErbB4 fusion protein pull down assay and mass spectroscopic analysis, we have found the ErbB receptors interact with nucleolin via their cytoplasmic tail. Nucleolin is a ubiquitous, nonhistone, nucleolar, multifunctional phosphoprotein that is also overexpressed in cancer cells. Our results demonstrate that overexpression of ErbB1 and nucleolin may lead to receptor dimerization, phosphorylation and to anchorage independent growth. CONCLUSIONS/SIGNIFICANCE: The oncogenic potential of ErbB depends on receptor levels and activation. Our results suggest that nucleolin may affect ErbB dimerization and activation leading to enhanced cell growth.