For stringent identification of differences across sam ples, phosphopeptides we

We found an additive effect of combining these com pounds in the viability of all these cell lines, Interestingly, the cell lines with greater concentrations of phosphorylated MARKCS were more resistant to the PKC inhibition relative Amuvatinib ic50 to those with low phospho MARCKS, These data are in line with previous findings suggesting that the PKC signaling pathway has a role in the proliferation and or survival of AML cell lines, We also found that while the intensities of phosphory lated STAT5A, a marker of JAK activities, did not corre late with the responses of our AML cell panel to JAK i, the intensities of phosphorylated Ribosomal S6 did corre late with resistance to this compound, As an example of a phosphoprotein that correlated with the responses to MEK i, Figure 7D shows the correlation of several phosphorylation sites on Stathmin, alongside the viability of cells exposed to this compound.<br><br> The phos phorylation of S16, S38, and S63 on Stathmin followed the same trend and had a relatively poor correlation with the response to MEK i compared to the phosphorylation of S25, a site that can be phos phorylated by several MAP kinases and cyclin depen dent kinases, Taken together, the data shown in AT-406 availability Figure 7 suggest that the activation of the pathway being targeted did not in general correlate with the response to kinase inhibitors and that phosphorylations that correlate with responses predominantly were on pathways parallel to those being targeted.<br><br> Phosphorylation motifs associated with responses to kinase inhibitors Phosphoprotein concentrations in cells can be a conse quence of the expression level of the phosphoprotein bearing the site of modification as well as of the activity of the AG-490 ic50 kinases and phosphatases acting on these sites. To investigate whether the phosphorylation sites that correlated with resistance or sensitivity in this study were the result of differential kinase activities across the AML cell line panel or these were just a reflection of phosphoprotein gene expression, we performed a further bioinformatics analysis of the data aimed at assessing common phosphorylation motifs in phosphopeptides correlating with the responses to the inhibitors. In this, phosphorylation motifs were obtained from the data using motif X and from the literature, The normalized intensities of phosphorylation sites bearing these motifs were then averaged and correlations with the viability of cells to each inhibitor recalculated for each of the averaged motif intensities.<br><br> This analysis was based on phosphopeptides with R 0. 45 or R 0. 45. Figure 8 shows that phosphorylation motifs that corre lated with resistance were rich in basic residues and or had a proline at the carboxyl terminus of the phosphory lated residue. In contrast, phosphorylation motifs that correlated with sensitivity to the inhibitors were predo minantly on acidic motifs for PI 103 and JAK i or they contained a hydrophobic residue at the N terminus for JAK i and MEK i, These data suggest that basophilic kinases and proline directed kinases may be more active in resistant cells, whereas acidophilic kinases and hydrophobic amino acid directed kinases may be more active in sensitive cells.