Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From

The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant () (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated . We further showed that autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity . This study provides insights into molecular basis for regulation of Stk1 activity from .