| Protein phosphorylation is most commonly exploited by cells to allow appropriate responses to various environmental cues, which plays an important role in cell signaling. However, in the Archaea, little is known regarding which proteins are phosphorylated and which kinases are involved. In this study, we have identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase (STPK) ST1565 and its protein partner, a forkhead-associated (FHA) domain-containing protein ST0829 from the archaeon Sulfolobus tokodaii str.7. We analyzed the reactive conditions of phosphorylation, and the critical amino acid residues in the conserved domain of both proteins, the major results were obtained as follows:(1) Using bacterial two-hybird assay and Pull-down/western experiments, the protein kinase ST1565 was shown to physically interact with the FHA domain protein ST0829. (2) Protein kinase ST1565 preferred Mn2+as its cofactor for auto-phosphorylation and for substrate-phosphorylation, at an optimal temperature 45℃and optimal of pH 5.5-7.5. (3) We analyzed and identified the critical amino acid residues in the conserved FHA and kinase domain sites through a series of mutation assays, and most mutional conservative amino acid residues lowered the activity of phosphorylation. Threonine-329 was suggested as a major activation site in the kinase. In contrast, Threonine-326 was a negative regulation site. (4) Several amino acid substitution mutants in the conserved FHA domain sites of ST0829 lost their physical interactions with ST1565, and the structure model for the FHA domain demonstrated that these mutation sites were located at the edge of the protein, and perhaps constituted the potential interaction domain with ST1565.This work presents pioneering work on the third domain of the Archaea, showing that a protein kinase interacts with and phosphorylates its FHA domain protein. These data provide critical information on the structural or functional characteristics of archaeal proteins and can accelerate the understanding of fundamental signaling mechanisms in all three domains of life forms. |
