Dataset Information

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus.

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 ?-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.

SUBMITTER: Zheng W

PROVIDER: S-EPMC5920020 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus.

Zheng Weihao W Cai Xiaodan X Li Shuiming S Li Zigang Z

Frontiers in microbiology 20180420

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 activatio ...[more]

PMID: 29731745

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Project description:Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the WHO as a human pathogen that urgently needs the development of new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has proven to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed a stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a Tandem Mass Tag (TMT) labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to the stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in ∆stk1 compared with P. aeruginosa PAO1. A detailed bioinformatics analysis of these differentially expressed proteins was performed, including GO annotation, protein domain profile, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localization and enrichment analysis. Notably, the downregulation of type IV pilus-related proteins and upregulation of T6SS-H1-related proteins were found in the ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens bacterial twitching motility and promotes a growth competition advantage, which are, respectively, mediated by type IV pilus-related proteins and T6SS-H1-related proteins. These findings contribute to a better understanding of the physiological role of Stk1, and proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, although the detailed regulation and mechanism of Stk1 still need to be revealed.

Project description:BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa) but given that Thr and Ser are not equivalent amino acids this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild-type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.

Dataset Information

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus.

Publications

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From <i>Staphylococcus aureus</i>.

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