Figure 3.
Adi3 phosphorylates Gal83 at Ser-26. A, Adi3 phosphorylates Ser-26 of Gal83. Kinase-active MBP-Adi3S539D was used to phosphorylate the indicated MBP-Gal83 Ser-to-Ala mutants using [γ-32P]ATP in in vitro kinase assays. Quantity One software was used to normalize the phosphorylation levels to the protein levels in each assay. Gal83 phosphorylation values are reported as a percentage of wild-type (WT) Gal83 phosphorylation and are averages of three independent experiments. Error bars represent se. Asterisks indicate significant increases (*) or decreases (**) in phosphorylation of Gal83 Ser-to-Ala mutants compared with wild-type Gal83 phosphorylation (Student’s t test, P < 0.05). Top and middle panels show phosphor images, and the bottom panel shows a Coomassie blue-stained gel. B, MS identification of Gal83 Ser-26 in vitro phosphorylation by Adi3. In vitro Adi3-phosphorylated Gal83-MBP as in A was digested with trypsin, passed over an immobilized metal affinity chromatography column, and eluted peptides were analyzed by MS/MS. C, MS identification of Gal83 Ser-26 in vivo phosphorylation. Gal83-GFP was expressed in tomato protoplasts, α-GFP was immunoprecipitated, the protein was digested with trypsin and passed over an immobilized metal affinity chromatography column, and the eluted peptides were analyzed by MS/MS. dN, Deaminated Asn; pS, phospho-Ser. [See online article for color version of this figure.]