Theileria annulata surface protein (TaSP) is a target of cyclin‐dependent kinase 1 phosphorylation in Theileria annulata‐infected cells

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour‐like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite‐dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA‐based experiments using a phospho‐threonine‐proline (pThr‐Pro) antibody revealed, that CDK1‐cyclin B specifically phosphorylates T131, identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata‐infected cells. Further studies demonstrated a nearly complete co‐localization of the pThr‐Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle‐dependent event. Immunostainings of isolated, non‐permeabilized schizonts confirmed the presence of the pThr‐Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr‐Pro signal of the schizont, which was reconstituted by the addition of CDK1‐cyclin B. Treatment of T. annulata‐infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin‐rich cell protrusions and an extension of the schizont, and a dose‐dependent reduction of BrdU incorporation and Ki67 staining of T. annulata‐infected cells, demonstrating a clear impact on the Theileria‐dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria‐infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.