The mammalian microtubule-associated serine/threonine (MAST) kinases are a highly conserved subfamily of AGC kinases that are implicated as therapeutic targets for cancer and diabetes. However, the activity, regulation, and substrates of MAST kinases are poorly understood. We examined the biochemical activity of Mast2, as a representative of the MAST family. The domain of unknown function (DUF1908) is necessary for Mast2 kinase activity in vitro, while the PDZ domain is dispensable. Mast2 kinase activity does not appear to be compatible with the AGC kinase model of T-loop phospho-activation. Instead, it contains a unique insertion that is likely stabilized by ion-pair interactions. The C terminus of the kinase domain contains motifs regulated by mechanistic target of rapamycin (mTOR) in other AGC kinases, and mutation of these conserved residues reduces Mast2 kinase activity. Consistent with mTOR regulation, Mast2 purified from insulin-stimulated cells has increased activity compared to serum-starved cells, and this increase in activity is dependent on mTOR. Finally, stable 18O-ATP labeled kinase assay linked phospho-proteomics identifies a collection of putative Mast2 substrates, including the PP2A inhibitor, endosulfine-α. Our results develop a biochemical profile of the MAST kinases, provide insight into their regulatory mechanisms, and begin to identify the cellular function of MAST2.
Keywords: AGC; AlphaFold; DUF 1908; ENSA; MAST1; MAST2; MAST3; MAST4; kinase; mTOR; phosphorylation; signal transduction.
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