Fig. 12: Distinct phosphorylation signatures in NIH3T3 cells expressing oncogenic Erk1^R84S and Erk1^R84H revealed by Phosphoproteomic analysis.

A Venn diagrams of pairwise comparisons of phosphor-proteomic data aimed at identification of phosphorylation sites modified specifically in NIH3T3 cells expressing Erk1^R84S or Erk1^R84H, compared to cells expressing Erk1^WT. Upper diagram: of 894 phosphorylation sites modified in day 1 in Erk1^R84S-transformed cells compared to cells expressing Erk1^WT and of 1187 sites modified in cells transformed by Erk1^R84H, 370 are common. Middle diagram: of 1220 sites specifically modified in day 3 in cells expressing Erk1^R84S and of 2319 sites modified in cells expressing Erk1^R84H, 611 are common. Lower diagram: of the 370 sites modified in day 1 specifically in clones expressing Erk1^R84S and Erk1^R84H and of the 611 sites specifically modified in day 3, 72 are common. B Heatmap of the 72 shared phospho-sites in cells expressing Erk1^R84S or Erk1^R84H on both days but absent in Erk1^WT, divided to 2 clusters of upregulated and downregulated phosphorylation in cells expressing Erk1^R84S or Erk1^R84H compared to cells expressing Erk1^WT. C Venn diagram of hierarchal pairwise comparisons of phosphor-proteomic data aimed at identification of differentially phosphorylated phosphosites between day 1 and day 3 of the culture’s life. Of 1546 sites differentially phosphorylated between day 3 and day 1 in cells expressing Erk1^R84S and of 3174 sites differentially phosphorylated in cells expressing Erk1^R84H, 691 are common. Of 1328 sites modified between day 3 and day1 in cells expressing Erk1^WT, 276 are common with sites modified in cells expressing Erk1^R84S or Erk1^R84H. Thus, 415 phosphoacceptors are differentially phosphorylated on day 3 vs day 1 in cells transformed by Erk1^R84S or Erk1^R84H and not in cells expressing Erk1^WT. D Heatmap of the 415 phospho-sites differentially phosphorylated on day 3 vs day 1 in the transformed cells, divided into six clusters as revealed by bioinformatics analysis.