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.
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Boyce Thompson Ibstitute for Plant Research and Department of Plant Pathology, Cornell University, Ithaca, New York 14853, USA.
D'ascenzo
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Martin
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D010748:Phosphopeptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; C084354:Pto protein, Lycopersicon; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin