br course Medians of all bins are connected to
course. Medians of all 10 bins are connected to indicate the shape of signaling relationships (black lines), with the relationship strength quantified by signed-BP-R2, as shown on top of each individual plot. In the plot on the far right, medians of each bin are connected over the time course to demonstrate the POI abundance-dependent signaling trajectories.
(H) Schematic illustration of how two sets of phosphatases induce different abundance-dependent influences on the signaling dynamics of the MAPK-ERK cascade.
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Different p-MEK1/2 dynamics were observed in FG4592 overex-pressing these two phosphatases. In response to the EGF stim-ulation, cells with DUSP4 overexpression had more sustained MEK1/2 activation (Figure 5F) compared to cells with PTPN2 overexpression (Figure 5G). DUSP4 has been shown to specif-ically target ERK1 and ERK2 (Guan and Butch, 1995). Our data indicate that by diminishing ERK1/2 phosphorylation, the overex-pressed DUSP4 attenuates the negative feedback from ERK1/2 to MEK1/2, resulting in constant activation of MEK1/2. Sub-strates of PTPN2 are primarily membrane kinases, including EGFR (Mattila et al., 2005). As expected, overexpression of PTPN2 downregulated the activation of all measured signaling proteins known to be downstream of EGFR, including MEK1/2 and ERK1/2 (Figure 5H).
To systematically classify all overexpressed phosphatases based on the signaling dynamics, we performed shape-based clustering on p-MEK1/2 trajectories during the 1-h time course after EGF addition, over the expression levels of each POI (Fig-ures S6E–S6H). Abundance-dependent prolonged MEK1/2 phosphorylation was observed with other phosphatases in group E, including DUSP6, DUSP7, DUSP10, DUSP16, and PTPN7, indicating that Thr202 and Tyr204 on ERK1/2 are sub-strates of these enzymes (Figure 5H). DUSP10 and DUSP16 have been previously reported to be JNK- and p38-specific phosphatases (Finch et al., 2012; Masuda et al., 2003). Here, we show that DUSP10 and DUSP16, expressed at high abun-dance, also dephosphorylate p-ERK1/2 and attenuate the MAPK-ERK signaling, thereby likely decreasing the negative feedback from ERK1/2 to MEK1/2 and causing sustained MEK1/2 activation (Figures 5A and 5H).
Pairwise Overexpression Analysis Reveals that Phosphatases Sustain Kinase-Induced MAPK-ERK Signaling
Phosphatase overexpression is oncogenic in different tumor types, but the signaling mechanisms remain unclear (Julien et al., 2007, 2011). Recent work indicates that overexpressed phosphatases increase the malignancy of cancers that have a hyperactivated MAPK-ERK pathway (Julien et al., 2007; Low and Zhang, 2016; De Vriendt et al., 2013). Our data suggest a mechanism through which overexpression of ERK-specific phosphatases sustains MEK phosphorylation levels (Figures 5F and 5H). To assess whether an additional, secondary signaling input that increases MAPK pathway activity could lead to phosphatase-driven oncogenic-like signaling, we developed a combinatorial transfection assay in which overexpression of a
kinase and a phosphatase were detected via an FLAG-tag and a GFP-tag, respectively, providing a two-dimensional analysis of abundance-dependent signaling modulations on the single-cell level (Figure 6A). Using this approach, we analyzed the MAP2K2, MAPK1, and RPS6KA1 (also known as MEK2, ERK2, and p90RSK1) kinases and the DUSP4, DUSP7, and PTPN2 phosphatases in 9 combinations of double overexpression over a 1-h EGF stimulation time course (Figure 6B).
When overexpressed individually, we observed that DUSP4 overexpression sustained the phosphorylation of Ser221 on MEK1/2 over the 1-h EGF stimulation time course, likely due to the weakened ERK-to-MEK negative feedback (Figures 6C and 6D). MAP2K2-FLAG overexpression led to an increased MEK1/2 phosphorylation (Figure 6C). MAP2K2-FLAG and DUSP4-GFP co-overexpression further increased the hyperactivated states of MEK1/2 over the 1-h EGF stimulation time course compared to the activation induced by MAP2K2-FLAG overexpression alone (Figures 6C–6E). Moreover, in cells with simultaneously overex-pressed MAP2K2-FLAG and DUSP4-GFP, the downstream ERK1/2 phosphorylation on Thr202 and Tyr204 were inhibited (Figures 6C–6E). Previously, highly activated MEK1/2 was observed to lead to ERK-independent oncogenic-like signaling (Burgermeister and Seger, 2008; Takahashi-Yanaga et al., 2004).
The overexpression of FLAG-tagged MAPK1 (ERK2) drasti-cally augmented ERK1/2 phosphorylation during EGF stimula-tion (Figure 6F), increased p-ERK1/2 amplitudes, and delayed p-ERK1/2 peak times (Figure 6G) in agreement with a previous study of the effect of MAPK1 overexpression (Lun et al., 2017). The simultaneous overexpression of MAPK1-FLAG and DUSP7-GFP decreased p-ERK1/2 levels at all time points and reduced the signaling amplitudes. Furthermore, DUSP7 overex-pression delayed p-ERK1/2 peak times upon EGF stimulation: in cells with the highest MAPK1 abundance and mid-level overex-pression of DUSP7, ERK1/2 phosphorylation peaked at 30 min after the addition of EGF (Figures 6F and 6G, purple arrows), whereas in untransfected cells, p-ERK1/2 peaked at the 5-min time point (Figures 6F and 6G). As expected, DUSP7 overex-pression also resulted in constant MEK1/2 phosphorylation (Figures 6F and 6G, green arrows). Compared to cells overex-pressing only MAPK1 (ERK2), which induced strong but transient ERK activation, the additional low-to-mid levels of DUSP7 decreased the ERK1/2 phosphorylation amplitude and partially limited the negative feedback signal from ERK to MEK, inducing a sustained MEK activation and a prolonged ERK signal. Thus, our analysis indicates that overexpression of certain phospha-tases, such as DUSP4 and DUSP7, led to sustained activation