Fig. 3.

Human kinome screen identified kinases involved in α_2A-adrenergic receptor (α_2A-AR) activation-mediated purinosome formation. (A) Characteristic DMR of HeLa cells in response to sequential stimulation steps (S1 and S2). Red line: buffer (S1) – buffer (S2); black line: buffer – TBB; purple line: EPI – buffer; blue line: EPI – TBB. The DMR of assay buffer stimulation was used as the negative control. Buffer by itself triggered little DMR, and did not alter DMR induced by 100 nM epinephrine (EPI). EPI (100 nM) triggered a positive DMR. Conversely, TBB led to a negative DMR in the buffer pretreated cells, but a much greater negative DMR in EPI pretreated cells. (B) The robust z-score of EPI-induced DMR (green dots) or TBB-induced DMR (red dots) as a function of shRNA clones. Robust z-scores (a z-score not adversely affected by outliers) were calculated using [(experimental data − median)/median absolute deviation (MAD)] where the normalization set the median to 0 and the MAD to 1. (C) Network analysis of the α_2A-AR activation mediated purinosome formation. This analysis combines all hits common to the EPI and TBB DMR responses identified using the current kinome screen with known signaling components of endogenous α_2A-AR in HeLa cells, casein kinase 2 (CSNK2B, CSNK2A1, CSNK2A2) and six enzymes (PPAT, GART, PFAS, PAICS, ADSL, ATIC) involved in purine biosynthesis. Hits were selected when at least two shRNA clones for a kinase within the library gave a robust z-score of ≥ 3 or ≤ −3 (Table S2). The network was generated using STRING 9.1. Connecting lines are color coded by the type of evidence used to build the network (details in http://string-db.org/). Unconnected hits are listed at the bottom. (D) Top panel: The real-time DMR of EPI in the absence (red) or presence of everolimus (green). Bottom panel: the real-time DMR of TBB after EPI pre-stimulation in the absence (red) or presence of everolimus (green). The dose was 16 μM, 100 nM, or 20 μM for everolimus, EPI, or TBB, respectively. For (A, D), data represents mean + standard deviation, N=4. The standard deviation is shown in gray.