Figure S5.
In vitro and in vivo evidence for TRAPPII phosphorylation by AtSKs. (A) In vitro kinase assays using GST:AtSK11 (72 kDa) and GST:TRS120-T2 (100 kDa). The change of phosphosignal is shown in a representative autoradiograph (upper panel) and the loaded protein amount in the corresponding CBB (Coomassie stain, lower panel). Non-phosphorylatable S to A TRS120-T2 variants were used as negative controls. The means ± SD of phosphosignals were normalized to the protein amount and related to non-mutated TRS120-T2 wild-type control. Note that AtSK11 phosphorylated AtTRS120-T2 in vitro, with a preference for wild-type (WT) sequences over non-phosphorylatable AtTRS120-SαβγA. n = 2–4 independent experiments; *: P < 0.05 for significant differences to TRS120-T2 WT (set at 1.0 right panel) determined by using a one sample two-tailed t test. (B) In vitro kinase assay with mass-spectrometry readout using BIN2 as kinase and TRS120-T2 truncation as substrate. Dilution series of the kinase are depicted in different shades of blue. BIN2 phosphorylated the β and γ site of TRS120 with a preference for the γ site (1e7 for TRS120-γ versus 1e6 for TRS120-β on the Y axis). Samples incubated for 120 min in a kinase buffer without ATP, or samples in which the kinase was heat-inactivated (KD), served as negative controls. The numbers in gray in each plot denote the number of times the phosphorylation event was seen in the given number of independent replicates. (C) Bikinin is an inhibitor of shaggy-like kinases (AtSKs), whereas PPZ is a BR biosynthesis inhibitor that relieves BR-mediated BIN2 inhibition. (D) Impact of the pharmacological inhibitors bikinin and PPZ on the TRS120 phosphorylation status in vivo. IP-MS was carried out on light-grown TRS120:GFP seedlings treated with bikinin, PPZ, or the respective mock-controls. The phosphorylated peptides were further analyzed via Skyline (MacLean et al., 2010). Normalized intensities were calculated as the ratio of the TRS120-Sγ phosphopeptide intensity over the sum of all TRS120 peptide intensities found in the respective experiment. The extent of phosphorylation at the TRS120-γ site was significantly decreased by bikinin. In contrast, PPZ treatment increased the phosphorylation of the TRS120-Sγ peptide in vivo. P values were computed with a two-tailed Student’s t test (*: P < 0.05; **: P < 0.01). Mean ± SD of four replicates are shown for the control and treatment. Related to Figs. 2, 3, and 4. Source data are available for this figure: SourceData FS5.

In vitro and in vivo evidence for TRAPPII phosphorylation by AtSKs. (A) In vitro kinase assays using GST:AtSK11 (72 kDa) and GST:TRS120-T2 (100 kDa). The change of phosphosignal is shown in a representative autoradiograph (upper panel) and the loaded protein amount in the corresponding CBB (Coomassie stain, lower panel). Non-phosphorylatable S to A TRS120-T2 variants were used as negative controls. The means ± SD of phosphosignals were normalized to the protein amount and related to non-mutated TRS120-T2 wild-type control. Note that AtSK11 phosphorylated AtTRS120-T2 in vitro, with a preference for wild-type (WT) sequences over non-phosphorylatable AtTRS120-SαβγA. n = 2–4 independent experiments; *: P < 0.05 for significant differences to TRS120-T2 WT (set at 1.0 right panel) determined by using a one sample two-tailed t test. (B) In vitro kinase assay with mass-spectrometry readout using BIN2 as kinase and TRS120-T2 truncation as substrate. Dilution series of the kinase are depicted in different shades of blue. BIN2 phosphorylated the β and γ site of TRS120 with a preference for the γ site (1e7 for TRS120-γ versus 1e6 for TRS120-β on the Y axis). Samples incubated for 120 min in a kinase buffer without ATP, or samples in which the kinase was heat-inactivated (KD), served as negative controls. The numbers in gray in each plot denote the number of times the phosphorylation event was seen in the given number of independent replicates. (C) Bikinin is an inhibitor of shaggy-like kinases (AtSKs), whereas PPZ is a BR biosynthesis inhibitor that relieves BR-mediated BIN2 inhibition. (D) Impact of the pharmacological inhibitors bikinin and PPZ on the TRS120 phosphorylation status in vivo. IP-MS was carried out on light-grown TRS120:GFP seedlings treated with bikinin, PPZ, or the respective mock-controls. The phosphorylated peptides were further analyzed via Skyline (MacLean et al., 2010). Normalized intensities were calculated as the ratio of the TRS120-Sγ phosphopeptide intensity over the sum of all TRS120 peptide intensities found in the respective experiment. The extent of phosphorylation at the TRS120-γ site was significantly decreased by bikinin. In contrast, PPZ treatment increased the phosphorylation of the TRS120-Sγ peptide in vivo. P values were computed with a two-tailed Student’s t test (*: P < 0.05; **: P < 0.01). Mean ± SD of four replicates are shown for the control and treatment. Related to Figs. 2, 3, and 4. Source data are available for this figure: SourceData FS5.

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