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Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via ␣ 5 ␤ 1 -or ␤ 3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110␥ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110␥. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110␥ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110␥ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.The Eph receptor tyrosine kinases (RTKs), together with their ephrin ligands, regulate diverse developmental patterning processes including axon guidance, cell migration, and cell segregation (13). However, in contrast to other families of receptor tyrosine kinases, the Eph RTKs do not appear to regulate cell proliferation and survival. It was recently reported that activation of the Eph RTKs by their cognate ligands leads to changes in cell adhesion to various extracellular matrix proteins. For example, EphB1 promoted cell attachment to fibronectin or fibrinogen, whereas neither a kinase-inactive EphB1 mutant nor EphB1 point mutants defective for binding to either Nck or low-molecular-weight protein tyrosine phosphatase (LMW-PTP) showed this effect (21, 35). EphB2 was also shown to indirectly control integrin activity by inducing tyrosine phosphorylation of R-Ras, possibly through a novel signaling intermediate, Src homology 2 (SH2) domain-containing Eph receptor binding protein 1 (SHEP1) (9, 43). More recently, EphA2 kinase was reported to regulate integrin function by causing focal adhesion kinase dephosphorylation (26). These results are consistent with the concept that the kinase activity of the Eph RTKs plays a pivotal role in regulation of ce...

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-␣ led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-␣-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways.