Autoregulatory characteristics of a Bacillus anthracis serine/threonine kinase.

ABSTRACT

BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.