Simultaneous detection of Akt and MAPK signaling pathway targets reveals reciprocal regulation of Phospho-Akt(S473) and Phospho-S6RP(S235/236 and S240/244) as a function of cell density

1680 Cell density is a potent regulator of the cell cycle during exponential growth and thus affects gene expression. Many recent studies have characterized cell-density as a controlling factor for cell-cell interactions and the binding of surface-associated adhesion molecules to the cytoskeleton. We examined the impact of cell density on two distinct signal transduction pathways, Protein kinase B (Akt) and Mitogen-activated protein kinases (MAPK). These pathways regulate cell proliferation, differentiation, and apoptosis, and exhibit cross-talk. Downstream, the MAPK and Akt pathways regulate p70S6kinase (p70S6K) and its substrate S6 ribosomal protein (S6RP). Whereas levels of phospho-S6RP are known to be regulated by phosphorylated-Akt, our findings suggest that in Jurkat cells this is dependent upon the cell density. We show that maximum phosphorylation of S6RP(S235/S236 and S240/244) is observed at lower cell densities (0.5x106 cells/mL). In contrast, levels of phosphorylated Akt increase with higher cell densities (1.3x106 cells/mL). In order to study the impact of cell density over a wide range of targets in the two pathways, we have developed highly sensitive multiplex assays that detect multiple phosphoproteins simultaneously in a single well with low microgram levels of cell lysate protein. Low and high density Jurkat cells were interrogated for the phosphorylation status in multiplex panels of S6RP/Akt/p70S6K/GSK3β and ERK/JNK/p38; the results from these studies will be presented.