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  • FG 4592 br Overexpression of a poorly

    2020-08-12


    Overexpression of a poorly characterized kinase, MGC42105 (NIM1K), in cluster 5 (highlighted in Figure 4D) modulated the phosphorylation of Ser241 on PDK1, Thr389 on p70S6K, and Ser235/Ser236 on S6, suggesting a role in growth signaling. MGC42105 also induced abundance-dependent activation of stress pathways, as strong relationships to p-p53 (Ser15) and p-AMPKa (Thr172) were observed (Figure 4D). In summary, mapping our identified signaling relationships to the OmniPath database enabled the assignment of signaling functions to a number of kinases and phosphatases and shed light on potential signaling mechanisms associated with the poor prognosis of cancer patients.
    In-Depth Analysis of Signaling Dynamics Reveals Overexpression-Dependent MAPK-ERK Activity
    An understanding of signaling dynamics is essential for identi-fying diseased signaling circuits within a network and in the pre-diction of drug efficiency (Du and Elemento, 2015; Hughey et al., 2010). We have previously shown that altering the FG 4592 levels of central signaling proteins in the EGFR signaling network results in complex changes in network dynamics (Lun et al., 2017). Given the key role of signaling dynamics on cell prolifera-tion, growth, and differentiation (Koseska and Bastiaens, 2017), we systematically evaluated kinases and phosphatases from the 10 identified clusters for overexpression-induced signaling dy-namic modulations. We calculated the differences in signed-BP-R2 scores between the EGF-stimulated and -unstimulated conditions to identify cases in which overexpression modulated signaling dynamics (i.e., altered the strength or the shape of abundance-dependent signaling relationships between the un-stimulated and the 10-min EGF-stimulated conditions). We found that POIs in clusters 1, 6, 7, 9, and 10 strongly modulated signaling network dynamics when overexpressed (Figure S5A). We then analyzed the overexpression effects of the top 39 dynamic-modulating POIs over a 1-h EGF stimulation time course. The dynamic responses of all of the measured phos-phorylation sites are shown in Figures 5A and 5B. Example
    signaling relationship shapes at each time point during the time course and the POI abundance-dependent signaling trajectories over the time course are shown in Figures 5C–5G. Features of the signaling amplitudes (see STAR Methods) are shown in Fig-ures S5F–S5H.
    Hierarchical clustering of the overexpression-induced EGFR signaling dynamics classified the 39 selected proteins into
    6 groups (Figures 5A and S5B). Signaling network responses for one representative kinase or phosphatase from each of the six identified groups are illustrated in Figure 5B. We showed that the network responses to EGF stimulation were highly spe-cific to cell lines (Figure S5C). In HEK293T cells, EGF stimulation strongly activated the MAPK-ERK signaling pathway and had weak effects on the AKT, PKC, STAT, or stress response path-ways (Figure S5C). Similarly, we observed that EGF stimulation primarily influenced the POI abundance-dependent dynamics of the MAPK-ERK signaling cascade rather than the AKT, PKC, and STAT pathways (Figure 5A). As the MAPK-ERK proliferative pathway is known to be involved in tumor progression and drug response, we focused our subsequent analyses on this pathway in HEK293T cells.
    Phosphorylation of Thr202/Tyr204 on ERK1/2 was elevated in cells with high levels of GFP-tagged EGFR in the absence of EGF stimulation (Figures 5B and 5C). These cells did not respond to EGF stimulation, indicating ligand-independent ERK activation (Figures 5C and S5G). Since the ligand-indepen-dent ERK activation is known as a cancer-driving mechanism (Guo et al., 2015), we next sought to systematically identify signaling proteins causing similar abundance-dependent ligand insensitivity. Applying shape-based clustering, we classified p-ERK1/2 signaling trajectories during the 1-h EGF stimulation time course, over the range of expression levels of each analyzed POI. We found that overexpression of kinases TYRO3, TEC, MST1R, MOS, MET, MAP3K8, FGF1R, and ABL1 also led to prolonged ERK1/2 activation (Figures S6A– S6D). These proteins have been previously shown to mediate oncogenic signaling (Duan et al., 2016; Johannessen et al., 2010; Paul and Mukhopadhyay, 2004; Salgia, 2017).
    In the absence of EGF stimulation, overexpression of phos-phatases DUSP4 and PTPN2 did not affect the levels of phos-phorylation in the MAPK-ERK pathway (Ser221 on MEK1/2, Thr202/Tyr204 on ERK1/2, or Ser380 on p90RSK) (Figures 5B, 5D–5G, S5D, and S5E). This suggests either a mechanism that compensates for phosphatase overexpression to maintain basal MAPK-ERK signaling or that the overexpressed phosphatases are inactive without EGF stimulation. Upon EGF stimulation, signaling dynamics on phosphorylation sites of the MAPK-ERK pathway were modulated by DUSP4 and PTPN2 in an abun-dance-dependent manner as negative signaling relationships to p-ERK1/2 and p-p90RSK were detected (Figures 5B, 5D, 5E, S5D, and S5E). DUSP4 or PTPN2 overexpression also resulted in reduced p-ERK1/2 and p-p90RSK amplitudes (Figure S5H).