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Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of AGC kinase family, possessing a unique non-conserved middle region. The mechanism of Mastl activation has been studied extensively in vitro. Phosphorylation of several residues were identified to be crucial for activation. These sites correspond to T193 and T206 in the activation loop and S861 in the C-terminal tail of mouse Mastl. To date, the significance of these phosphosites was not confirmed in intact mammalian cells. Here, we utilize a genetic complementation approach to determine the essentials of mammalian Mastl kinase activation. We used tamoxifen-inducible conditional knockout mouse embryonic fibroblasts to delete endogenous Mastl and screened various mutants for their ability to complement its loss. S861A mutant was able to complement endogenous Mastl loss. In parallel, we performed computational molecular docking studies to evaluate the significance of this residue for kinase activation. Our in-depth sequence and structure analysis revealed that Mastl pS861 does not belong to a conformational state, where the phosphoresidue contributes to C-tail docking. C-tail of Mastl is relatively short and it lacks a hydrophobic (HF) motif that would otherwise help its anchoring over N-lobe, required for the final steps of kinase activation. Our results show that phosphorylation of Mastl C-tail turn motif (S861) is dispensable for kinase function in cellulo.
Erguven et al. (Fri,) studied this question.