These results argue that the observed differences in Akt activation between highand low density cells can’t be explained by differences in Alogliptin selleck kinase affiliation with upstream activators. suppressed relative to low density cells, the scale of EGFR activation in high density cells seems sufficient to fully activate the EGF dependent Erk1 2 process. Why does occurrence dependent reduction of EGFR activity keep the EGF dependent Erk1 2 pathway unaffected while suppressing the EGF dependent Akt pathway We examined the tyrosine phosphorylation states of EGFR substrates associated with Gab1, Akt activation and erbB3, to start to answer that question. Both erbB3 and Gab1 present EGF dependent increases in tyrosine phosphorylation. The Gab1 tyrosine phosphorylation had similar kinetics under both culture conditions and was maximal by 5 min. The EGF stimulated erbB3 tyrosine phosphorylation was maximal by 5 min, and remained basically unchanged under both density conditions throughout the EGF time program. Gab1 and erbB3 people were related under the low and high density conditions. These results indicate that the decreased EGF dependent Akt activation in high density cells is not merely a direct representation of the decreased EGFR activation in these cells. The reduced steady state EGFR activation in the highdensity cells doesn’t reduce signaling through the Erk1 2 route or even to Gab1 and erbB3. For that reason, EGFR signaling in high-density Mitochondrion cells, when it comes to its ability to activate downstream proliferative paths, is not inhibited. The important point of inhibition of EGF dependent growth in high density cells has to be downstream from the EGFR approximately Akt and Gab1 erbB3. It is a completely new finding and is a new type for contact inhibition of EGF dependent growth. Following tyrosine phosphorylation of Gab1 and erbB3, the next step in the EGF dependent activation of Akt is PI3 kinase activation. PI3 kinase is activated through organization of its p85 subunit with phosphotyrosine residues on Gab1 and erbB3. Do high density intercellular contacts inhibit Akt activation by inhibiting PI3 kinase activation Gab1 erbB3 and natural product libraries were immunoprecipitated, and the levels of p85 associated with these proteins were determined by Western blot analysis. Similar quantities of p85 were connected with Gab1 in the high and low density cells. EGF treatment triggered similar levels of erbB3 related p85 at both densities. The Gab1 associated PI3 kinase activation was measured by an in-vitro kinase assay to verify the number of p85 subunit associated with Gab1 reflects PI3 kinase enzymatic activity. No big difference in Gab1 linked PI3 kinase activation was observed involving the high and low density cells.