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  • Exendin 4 br We cloned open reading frames ORFs from

    2020-08-12


    We cloned open reading frames (ORFs) from the human kinase library (Johannessen et al., 2010) and the human phosphatase li-brary into a vector, enabling the expression of GFP-tagged proteins (Couzens et al., 2013). The generated 541 kinase and 108 phosphatase expression clones (Table S1) were individually 
    transiently transfected into human embryonic kidney HEK293T cells. Unstimulated Exendin 4 and cells stimulated for 10 min with EGF were harvested and processed with a 126-plex barcoding strategy (adapted from Bodenmiller et al., 2012; Zunder et al., 2015) for simultaneous antibody staining followed by multiplexed mass cytometry measurements (Figure 1A).
    Transient transfection generates a single-cell gradient of the GFP-tagged protein of interest (POI) expression levels with up to 1,000-fold enhancement relative to the endogenous POI expression range (Lun et al., 2017). The abundance variation of each overexpressed kinase or phosphatase was quantified on the single-cell level by mass cytometry with detection by a metal-conjugated anti-GFP antibody. Simultaneously, we quanti-fied 30 phosphorylation states of proteins involved in key cancer-related signaling pathways, including the AKT, protein kinase C (PKC), signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK)/ERK, and stress path-ways, and 5 non-signaling markers indicative of cell physiological states (Table S2). Over 10 million individual cells were analyzed in the 659 overexpression conditions with or without 10-min EGF stimulation, averaging 7,000 measured cells per sample. That the GFP-tagging system rarely influenced protein overexpression effects has been previously reported (Lun et al., 2017).
    The dependence of phosphorylation levels on the abundance of GFP-tagged POI was quantified by the binned pseudo-R2 (BP-R2) method, a density-independent measure of signaling relationship strength (Figures S1A and S1B) (Lun et al., 2017). We confirmed that signaling relationships assessed with our approach were reproducible in five different cell lines from multi-ple tissues of origin (Figure S1C). We analyzed 108 control sam-ples (FLAG-GFP overexpression or untransfected cells) and used the highest BP-R2 score (0.13) of all of the controls as the cutoff to consider a signaling relationship as ‘‘strong.’’ In total, our human kinome and phosphatome analysis detected 1,323 pairs of strong relationships between POIs and phosphorylation sites (Figure 1B). Among the 649 kinases and phosphatases, 327 (50.4%) had at least 1 strong signaling relationship to the cancer-related signaling network when overexpressed. Of these, 245 had narrow influences with the modulation of 1–5 signaling nodes, and 26 overexpressed proteins had broad effects on the network with >10 measured phosphorylation sites influenced (Figure S1D). We identified 49 proteins that affected all of the measured signaling pathways, including 11 receptor proteins (e.g., MET, fibroblast growth factor receptor 1 [FGFR1], and platelet-derived growth factor receptor A [PDGFRA]) and many MAPK cascade activators (e.g., MAP4K1, MAP4K2, and MAP4K5) (Figure 1C).
    To characterize how POI abundance modulates intracellular signaling, we performed shape-based clustering (see STAR Methods) on all of the detected strong signaling relationships. We classified these relationship shapes into 10 shape clusters (Figure 1D). Shapes 1–5 involve overexpression-induced signal upregulation, with sensitivity to abundance changes increasing from shape 1 to shape 5. Shape 6 is non-monotonic signaling re-lationships, as phosphorylation levels initially increase and then decrease as a function of POI abundance. Shapes 7–10 reflect overexpression-induced signaling downregulation, with sensi-tivity to POI abundance changes increasing from shape 7 to shape 10 (Figure 1D). Overexpression of a particular POI can
    A
    D
    Figure 1. Kinome- and Phosphatome-wide Screen for Effects of Protein Abundance on Signaling States and Dynamics
    (A) The experimental workflow: ORFs of 541 human kinases and 108 human phosphatases were cloned into a vector encoding GFP-tagged fusion proteins upon transient transfection into HEK293T cells. Cells with or without 10-min EGF stimulation were harvested, barcoded, and stained with antibody mix before mass-cytometry-based single-cell analysis.
    (B) Plot of counts versus BP-R2 values for control and experimental samples. Cutoff value was determined by analysis of the BP-R2 values in all of the control samples. Square-root transformation was applied on the y axis.
    affect different phosphorylation sites with dissimilar shapes of relationship (Table S3). We also found that 250 pairs of signaling relationships had different shapes before and after EGF stimula-tion, indicating that POI levels determine the strength of the signaling response to EGF stimulation.