The major site of the Pti1 kinase phosphorylated by the Pto kinase is located in the activation domain and is required for Pto-Pti1 physical interaction

The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two k inases physically interact in the yeast two-hybrid system, and Pto specific ally phosphorylates Pti1 in vitro. In this study, we identified and charact erized the major Pti1 site phosphorylated by Pto. Pto was expressed in Esch erichia coli as a maltose-binding fusion protein (MBP-Pto), and used to pho sphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S- transferase (GST-Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST-Pti1(K96N) was partially purified by rever se-phase HPLC and analyzed by matrix assisted laser desorption/ionization m ass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230-234. By ph osphoamino acid analysis, Thr233 was determined to be the phosphorylation s ite of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phospho rylation by MBP-Pto and Pti1 autophosphorylation, providing evidence that t he same Pti1 site is involved in the two reactions. Moreover, phosphorylati on of Thr233 appeared to be required for Pto-Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the int eraction between Pto and Pti1 in the yeast two-hybrid system.