Abstract
The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.
Highlights
The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle
Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the non-conserved middle region (NCMR), display catalytic activity and different targets, establishing a possible link with truncated mutations observed in cancer-related databases
By comparing the MASTL sites arising from autophosphorylation and after phosphatase treatment, we found that the autophosphorylation of 5 sites (S213, S552, S703, T847, and S875) and the action of external kinases in 6 residues, mainly in the NCMR, (S216, S303, S384, S556, S572, and S660) is determinant for kinase activity
Summary
MASTL is a unique kinase because of a non-conserved insertion of 550 residues within its activation loop. MASTL contains a unique long insertion of about 550 non-conserved amino acids between the kinase subdomains VII and VIII, which is the typical location of the activation loop [3, 15] Some studies have addressed MASTL activation [15, 16] and structural details in the absence of the NCMR region are available [26]; the kinase activation mechanism and the role of the NCMR remain unclear In this manuscript, we perform a combined analysis indicating that the NCMR region is essential for MASTL specificity and allosterically regulates the enzyme catalysis. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveal MASTL dynamics
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