Complementation cloning identifies the essentials of mammalian Mastl kinase activation

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of the AGC kinase family, possessing a unique non-conserved middle region (NCMR). The mechanism of its activation prior to mitosis has been extensively studied in Xenopus egg extracts. These studies found several residues (corresponding to T193 and T206 in the activation loop, and S861 in the C-terminal tail, i.e., C-tail of mouse Mastl) whose phosphorylations are crucial for enzymatic activation. To date, the significance of these phosphosites was not confirmed in live mammalian cells. Here, we utilize a complementation cloning approach to determine the essentials of mammalian Mastl kinase activity. We employed a tamoxifen-inducible conditional knockout system to delete the endogenous Mastl in mouse embryonic fibroblasts (MEF) and screened various mutants for their ability to complement its loss. MEFs, ectopically expressing different phosphorylation site mutants, were induced to undergo recombination-mediated knockout in their endogenous Mastl loci. S861A and S861D mutants were able to complement endogenous Mastl loss with proliferation rates comparable to WT. In parallel, we examined the available protein kinase structures having a phosphorylated C-tail. Among the published states, two distinct positionings of the C-tail phosphoresidue were observed. Energetic analysis of these states revealed that only one conformation highly contributes to the C-tail docking. Our in-depth sequence and structure analysis showed that Mastl pS861 does not belong to the conformational state, where the phosphoresidue contributes to the C-tail docking. The C-tail of Mastl is relatively short and it lacks the hydrophobic (HF) motif. In other AGC kinases, the C-tail phosphosite aids the anchoring of this motif over the N-lobe, leading to the final step of kinase activation. Together with the lack of HF motif in Mastl, our results suggest that phosphorylation of the C-tail turn motif phosphosite (S861) is auxiliary and is dispensable for mammalian Mastl kinase function. Furthermore, we demonstrated that complementation cloning is a powerful approach for screening the determinants of an essential protein’s functioning. ### Competing Interest Statement The authors have declared no competing interest.