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.

Bacillus anthracis chain length, a virulence determinant, is regulated by a transmembrane Ser/Thr protein kinase PrkC

“…2A and 2B). This result was in contradiction with 222 the earlier reports wherein B. anthracis prkC disruption was shown have no effect on the 223 bacterial growth in vitro (Shakir et al, 2010;Bryant-Hudson et al, 2011;Arora et al, 2017). 224…”

The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence

“…Additionally, we expected to observe phosphorylation in the putative activation loop, as this has been observed for the PASTA kinases in M . tuberculosis and Bacillus anthracis [58,59]. Although we did observe a quadruply phosphorylated fragment ion that contained the putative loop region, the large size of the fragment ion prevented our ability to map specific phosphorylation sites.…”

Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis

“…The blots were developed using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) according to the manufacturer's instructions. Autophosphorylated PrkC was kept as a negative control for the ␣-Tyr(P) blot because it has been reported to be phosphorylated on Ser and Thr residues (30). Phosphorylated myelin basic protein (MyBP) phosphorylated by the Tyr kinase Abl (New England Biolabs) was used as a positive control.…”

“…PrkC is the only characterized STPK in B. anthracis and B. subtilis (28,29). Recent data showed that the loss of this enzyme (called BA-Stk1) in B. anthracis had profound effects on virulence (29,30). Furthermore, it was found that germinating spores of B. subtilis use degraded peptidoglycan fragments called "muropeptides" as a germination signal, which is sensed by C-terminal penicillin-binding protein and Ser/Thr kinaseassociated (PASTA) domains of PrkC (31).…”