Because of the previous implication of other PI3Ks in eEF2 activity, we focused our attention on this protein. Enlarged regions of images and three dimensional fluorescence in tensity profiles of a representative spot 101 identified as eEF2 is shown in Figure 4A. All biological replicates showed a similar increase in abundance. The characteristics of the abundance of inhibitor supplier this protein as well as the short stimulation time used in the experiment strongly suggest that the difference observed is due to a posttransla tional modification, and based on the shift from the acidic to basic site of the gel, it is likely that the protein is less phosphorylated in the p110�� knockdown cell line when compared to the control cells.

Phosphorylation of eukaryotic Elongation factor 2 in response to IGF I is PI3K�� dependent To confirm the involvement of the regulation of eEF2 downstream of PI3K��, control and PI3K�� knock down cells were stimulated with IGF I and Inhibitors,Modulators,Libraries the lysates were immunoblotted with anti phospho eEF2, followed by stripping and reprobing of the western blot with anti eEF2 antibody. The results demonstrated that the phos phorylation of eEF2 was decreased in p110�� knockdown cells compared to that in the control cells, whereas the total eEF2 protein was not affected. Although the phosphorylation of eEF2 was initially shown to be significantly different after 5 min of Inhibitors,Modulators,Libraries stimulation, the response seems to be more re producible and robust after 10 min of stimulation. In addition, the lysates from parental MDA MB 231 cells pre treated with the isoform specific inhibitor, AS605240, followed by stimulation with IGF I were immunoblotted with phospho eEF2 and total eEF2 anti bodies.

The results of these experiments showed that the level of phospho eEF2 in response to IGF I was signifi cantly decreased after AS605240 treatment compared with control cells. Taken together, these data indicate that eEF2 is phosphorylated downstream of activation of the IGF 1R CXCR4 heterodimer in response Inhibitors,Modulators,Libraries to IGF I and that this is dependent Inhibitors,Modulators,Libraries on PI3K�� activation. Discussion Activation of receptor tyrosine kinases and G protein coupled receptors GPCRs by their ligands leads to the activation of intracellular signaling cascades. While these pathways were initially thought to be distinct, recent data indicate an important role for RTK GPCR transactivation in a number of physiological and patho logical cellular responses.

This form of receptor transactiva tion has been shown to regulate cell proliferation, migration and invasion in various types of can cer, and our recent data Inhibitors,Modulators,Libraries indicate an important role for IGF 1R and CXCR4 transactivation in migration of MDA MB 231 breast cancer cells. This IGF 1R CXCR4 heterodimer appears no to be linked with the metastatic phenotype of these cells as the related but non metastatic MCF 7 breast cancer cell line does not express functional heterodimers.