Fig. 3.

Subclass III SnRK2s directly phosphorylate Raf36. (A) In vitro phosphorylation assay using kinase-dead forms of GST-SRK2E (SRK2E K50N) or MBP-Raf36 (Raf36 K234N). Each kinase-dead form was coincubated with an active GST-SRK2E or MBP-Raf36 kinase as indicated. Assays were performed in the presence of 5 mM Mn²⁺ (Left three lanes) or 5 mM Mg²⁺ (Right three lanes) with [γ-³²P] ATP. (B) In vitro phosphorylation assay using truncated forms of MBP-tagged Raf36. Each MBP-Raf36 protein was incubated with MBP-SRK2E in the presence of 5 mM Mg²⁺ with [γ-³²P] ATP. N: N-terminal region, KD: kinase domain, C: C-terminal region. (C) Schematic representation of six Raf36 (134 to 163) tested as SRK2E substrates. Ser¹⁴¹, Ser¹⁴⁵, Ser¹⁵⁰, and Ser¹⁵⁵ are labeled in blue, with alanine substitutions shown in red. Ser¹⁵⁷, labeled in green, was replaced with alanine in Raf36 (134 to 163) #1 to #5. (D) In vitro phosphorylation of GST-Raf36 (134-163) #1 to #6 by MBP-SRK2E. (E) In vitro phosphorylation of GST-Raf36 (134-163) #3 by MBP-SRK2D or MBP-SRK2I. Autoradiography (³²P) and Coomassie brilliant blue (CBB) staining show protein phosphorylation and loading, respectively. (F and G) Phosphorylation of MBP-Raf36 Ser¹⁴⁵ identified by phosphopeptide mapping. The amino acid sequences with probable phosphorylated serine residue (pS), ion-current chromatograms (F), and MS/MS spectrum (G) derived from a phosphopeptide of m/z = 437.88+++ are shown. MBP-Raf36 protein was either phosphorylated by MBP-SRK2E (+ SRK2E) or autophosphorylation (Auto-P) for 30 min. (H) Phosphorylation levels of Raf36 Ser¹⁴⁵ (Upper) and Ser¹⁰¹ (Lower) after ABA treatment. Protein extracts were prepared from 2-wk-old Raf36pro:Raf36-3xFLAG/raf36-1 transgenic seedlings, which were treated with or without 50 µM ABA for 30 min, and total proteins were subjected to immunoprecipitation using anti-DYKDDDDK tag antibody beads followed by tryptic digestion. Peptides were analyzed by LC-MS/MS, and Raf36 phosphopeptides were identified and quantified based on the peak area of extracted ion chromatogram (XIC). Data are means ± SE from five independent biological replicates. Asterisks indicate significant differences (**P < 0.01, two-tailed Student’s t test). n.s., not significant.