Extended Data Fig. 8: UGDH–HuR interaction requires MEK1-dependent pUGDH(Y473).

a, A549 cells were treated with EGF (100 ng ml⁻¹) for 30 min. HuR was immunoprecipitated and treated with or without RNase (10 ng μl⁻¹) for 30 min. b, A549 cells were infected with or without the lentivirus expressing HA–HuR or Flag–UGDH and then treated with EGF (100 ng ml⁻¹) for 30 min. The immunoprecipitated complex was treated with or without calf intestinal alkaline phosphatase (CIP) for 30 min. c, A549 cells stably expressing Flag–UGDH were treated with or without EGF (100 ng ml⁻¹) for 30 min. d, e, Mass spectrometry analyses of UGDH-associated proteins were performed in A549 cells stably expressing Flag–UGDH treated with or without EGF (100 ng ml⁻¹) for 30 min. d, All phosphorylated tyrosine residues are listed. The precursor ion was fragmented by collision-induced dissociation and analysed using an ion trap. The database search engine (Andromeda) score was matched to the identified peptide. e, Mass spectrometry analyses of phosphorylation of Y108 in 103-AADLKpYIEACAR-114, phosphorylation of Y352 in 340-KDTGDTRESSSIpYISK-355, and phosphorylation of Y473 in 471-IPpYAPSGEIPK-481 were performed. The parameters including scan number, method, score and m/z are shown. f, A549 cells stably expressing Flag–UGDH(WT), Flag–UGDH(Y108F), Flag–UGDH(Y352F) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml⁻¹) for 30 min. g, H1299 cells stably expressing Flag–UGDH(WT) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml⁻¹) for 30 min. h, A549 cells (left) or A549 cells stably expressing Flag–UGDH(WT) (right) were treated with or without TGFβ (20 ng ml⁻¹) for 30 min. i, A549 cells stably expressing Flag–UGDH were pretreated with or without the MEK inhibitor U0126 (0.25 μM) for 4 h or the p38 inhibitor BIRB 796 (1 μM) for 4 h, followed by treatment with or without EGF (100 ng ml⁻¹) for 30 min. j, A549 cells were treated with or without the MEK inhibitor U0126 (0.25 μM) for 4 h (top) or the p38 inhibitor BIRB 796 (1 μM) for 4 h (bottom). k, A549 or H1299 cells stably expressing Flag–UGDH were infected with or without the lentivirus expressing shMEK1. The expression of MEK1 in the cells was examined (left). Cells were treated with or without EGF (100 ng ml⁻¹) for 30 min (right). l, A549 cells stably expressing shNT or shUGDH were treated with EGF (100 ng ml⁻¹) for 30 min. m, A549 cells stably expressing Flag–UGDH(WT) or Flag–UGDH(Y473F) were treated with or without EGF (100 ng ml⁻¹) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without purified recombinant His–RRM1/2. n, Flag–UGDH(WT) or Flag–UGDH(Y473F) were immunoprecipitated to determine UGDH enzymatic activity. Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test). a–c, f–n, Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments.

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