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 kinases physically interact in the yeast two-hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose-binding fusion protein (MBP-Pto), and used to phosphorylate 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 reverse-phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230-234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP-Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation 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 interaction between Pto and Pti1 in the yeast two-hybrid system.