Figure 4.
In vitro AtSK kinase assays with TRS120 as bait. (A) In vitro kinase assays using GST:AtBIN2 (69 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 BIN2 phosphorylated AtTRS120-T2 in vitro, with a preference for wild-type (WT) sequences over non-phosphorylatable AtTRS120-SγA, AtTRS120-SαγA, AtTRS120-SβγA and AtTRS120-SαβγA substrates. n = 3 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) Kinase assays were performed in vitro with mass-spectrometry readout. One member of each shaggy-like kinase clade (AtSKs; see Fig. S3 A) was used with a constant concentration of the TRS120-T2 truncation as substrate. The dilution series of the kinase are depicted in different shades of blue. AtTRS120-T2 has highly (red) and moderately (orange) conserved sequences, as well as plant-specific sequences (green). Three GSK3 sites (referred to as α, β, γ; see Fig. 2 C) can be found in the plant-specific T2 domain. AtSKs in clades I-III differentially phosphorylated the substrate at three GSK3 consensus sites (with a preference for the γ site) in a time-dependent and concentration-dependent manner. A clade IV AtSK did not phosphorylate at all. 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 grey in each plot denote the number of times the phosphorylation event was seen in the given number of independent replicates. Note the higher intensity of the TRS120-Sγ peptide, especially for Clade I/SK11 (1e8 for TRS120-γ versus 1e6 for TRS120-α and TRS120-β on the Y axis). Related to Figs. S2, S3, and S5. Source data are available for this figure: SourceData F4.

In vitro AtSK kinase assays with TRS120 as bait. (A) In vitro kinase assays using GST:AtBIN2 (69 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 BIN2 phosphorylated AtTRS120-T2 in vitro, with a preference for wild-type (WT) sequences over non-phosphorylatable AtTRS120-SγA, AtTRS120-SαγA, AtTRS120-SβγA and AtTRS120-SαβγA substrates. n = 3 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) Kinase assays were performed in vitro with mass-spectrometry readout. One member of each shaggy-like kinase clade (AtSKs; see Fig. S3 A) was used with a constant concentration of the TRS120-T2 truncation as substrate. The dilution series of the kinase are depicted in different shades of blue. AtTRS120-T2 has highly (red) and moderately (orange) conserved sequences, as well as plant-specific sequences (green). Three GSK3 sites (referred to as α, β, γ; see Fig. 2 C) can be found in the plant-specific T2 domain. AtSKs in clades I-III differentially phosphorylated the substrate at three GSK3 consensus sites (with a preference for the γ site) in a time-dependent and concentration-dependent manner. A clade IV AtSK did not phosphorylate at all. 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 grey in each plot denote the number of times the phosphorylation event was seen in the given number of independent replicates. Note the higher intensity of the TRS120-Sγ peptide, especially for Clade I/SK11 (1e8 for TRS120-γ versus 1e6 for TRS120-α and TRS120-β on the Y axis). Related to Figs. S2, S3, and S5. Source data are available for this figure: SourceData F4.

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