Fig 1: RKIP reprograms tumors by reducing signaling capacity of a network instead of targeting a single node.(A) Multiplexed inhibitor beads – mass spectrometry (MIB-MS) analysis of n = 5 control and n = 6 RKIP-expressing BM1 tumors, showing 23 kinases with reduced activity and seven kinases with enhanced activity by exogenous RKIP expression. Student’s t-test, p<0.05. (B) Kinome tree displaying the distribution of kinases targeted by RKIP across different families of kinases. Blue: activity reduced by RKIP (n = 23), Red: activity enhanced by RKIP (n = 7). (C) Gene set enrichment analysis of the 23 negatively regulated kinases by Metascape. Stress-induced mitogen activated protein kinase (MAPK) related gene sets are highlighted in red. (D), Ingenuity Pathway Analysis (IPA) of the RKIP target kinases centered around MAPKs p38, JNK, and ERK. (E) Direct protein-protein interaction network and community analysis showing the core of the RKIP kinase network. (F) Diagram summarizing the interactions within the RKIP-regulated stress MAPK network in anisomycin-induced BM1 cells. Kinase interactions are determined by using small molecule inhibitors or siRNAs against the kinases in the network in three or more independent dose-response experiments with similar results (also see Figure 2—source data 2). The TAOK-p38 interaction is observed in cells treated with a cocktail of siRNAs against all three TAOKs (siCombo), whereas TAOK-JNK and TAOK-ERK interactions were observed by siRNAs against TAOK1 and TAOK2, respectively. For the source data, see Figure 2—source data 1.Figure 2—source data 1.Source data for the MIB-MS analysis of RKIP-expressing BM1 xenograft tumors.Figure 2—source data 2.RKIP-regulated MAPK network displays extensive cross-talk and feedback.(A) Western blots from three independent experiments showing the effect of p38i, JNKi, and MEKi on the phosphorylation of known direct targets of p38, JNK, and MEK, respectively. To monitor the substrate phosphorylation of p38, JNK, and MEK under treatment, we chose their known substrates MAPKAPK2, c-Jun, and ERK1/2, respectively. Statistical test was performed by student’s t-test for each dose of the inhibitors with respect to the non-treated control sample. (B) Western blots from three or more independent experiments showing the crosstalk and feedbacks that exist within the BM1 stress-kinase MAPK network. The diagrams on the far left show the specific direct/indirect interaction between two kinase nodes of the network, determined by inhibition of one of the kinases with a small molecule inhibitor or siRNAs and monitoring the activity of the other kinase. For this analysis, we used canonical phosphorylation sites of each kinase that correlate with their activity based on literature. The results of these experiments were used to build the network topology for BM1 cells depicted in Figure 2F.