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Abstract
The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (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 in vivo phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans 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 in vivo 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 in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus.
Highlights
Staphylococcus aureus (S. aureus) is one of the major human pathogens that greatly impacts individuals and causes a variety of illnesses ranging from minor skin infections to life-threatening diseases, such as endocarditis, pneumonia, septicemia, and toxic shock syndrome (Lowy, 1998)
The common regulatory mechanism for activity of Ser/Thr kinases is the phosphorylation of a canonical kinase segment known as the activation loop, which is flanked by the conserved N-terminal DFG and C-terminal APE motifs
In order to identify the phosphorylation sites on activation loop of Stk1 in S. aureus, we conducted a phosphoproteomic study of S. aureus Newman strain
Summary
Staphylococcus aureus (S. aureus) is one of the major human pathogens that greatly impacts individuals and causes a variety of illnesses ranging from minor skin infections to life-threatening diseases, such as endocarditis, pneumonia, septicemia, and toxic shock syndrome (Lowy, 1998). Bacteria have evolved multiple signal transduction systems to sense the environmental stimuli including nutrient concentrations and oxygen tension, eliciting appropriate activation or inactivation of response regulators (Rakette et al, 2012; Wright and Ulijasz, 2014). This is generally achieved through reversible protein phosphorylation mediated by protein kinases/phosphatase pairs, including well-known bacterial signaling cascades of two-component systems (TCSs). Our findings comprise a step toward understanding the activation of RD-family kinases of eSTKs
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