Fig. 5: SAPK2 T162/T283 determines ABI5 phosphorylation.

a Verification of SAPK2-mediated phosphorylation of ABI5 using bacterial kinase assays (left) and in vitro phosphorylation assays (right). b Inhibition of SAPK2-mediated phosphorylation of ABI5 by PP2C30/49/51. CBB staining confirmed the presence of co-expressed PP2Cs-GST and SAPK2-His proteins alongside ABI5-GST. c Partial peptide map of SAPK2 autophosphorylation sites identified by LC-MS/MS, showing phosphorylation at T159, T162, and T283. Additional mass data for other sites were shown in Supplementary Fig. 15. d Validation of SAPK2 kinase activity at ten identified autophosphorylation sites. Mutants m1–m5 correspond to S12A, S109A/Y113A/S116A, T159A/T162A, T212A/T214A/S218, and T283A, respectively. e Effects of mutations at distinct SAPK2 autophosphorylation sites on its ability to phosphorylate ABI5 among these, T162/T159 and T283 were critical for ABI5 phosphorylation, alanine substitution at these residues abolished SAPK2-mediated phosphorylation of ABI5. f Analysis of the impact of T159 and T162 mutations on SAPK2 autophosphorylation, demonstrating that T162, but not T159, was essential for SAPK2 autophosphorylation. g–i Effects of SAPK2 autophosphorylation site mutations on its interaction with ABI5. Co-IP (g), SLC(h), and in vitro pull-down (i) assays showed that mutations at T283 disrupted the SAPK2-ABI5 interaction, while mutations at other sites did not significantly alter interaction strength. Calf intestinal alkaline phosphatase (CIAP) was used as a phosphatase control in (a, d). Phos-tag gels were used to detect phosphorylated proteins in (a, b, d, e), and α-Ser/Thr immunoblotting was performed in (a, d–f) to assess phosphorylation status. In (i), “PD” indicates pull-down assay. Experiments shown in (a, b, d, e, f, g, h, and i) were repeated three times, yielding similar results. Source data are provided as the Source Data file.