Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis: IDENTIFICATION OF THE FIRST PROKARYOTIC DUAL SPECIFICITY TYROSINE PHOSPHORYLATION-REGULATED KINASE (DYRK)-LIKE KINASE

Gunjan Arora,Andaleeb Sajid,Mary Diana Arulanandh,Anshika Singhal,Abid R Mattoo,Andrei P Pomerantsev,Stephen H Leppla,Souvik Maiti,Yogendra Singh

doi:10.1074/jbc.m112.351304

Abstract

Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation.

Highlights

Characterization of Ser/Thr protein kinases present in Bacillus anthracis genome
PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates
Detailed experimental analysis showed that both kinases belong to separate classes of dual specificity protein kinases, with PrkD being the first bacterial “dual specificity tyrosine phosphorylation-regulated kinase” (DYRK)

Summary

Background

Characterization of Ser/Thr protein kinases present in Bacillus anthracis genome. Results: Dual specificity protein kinases were identified, of which one is similar to the eukaryotic DYRK superfamily. The guanidine phosphotransferase domain-containing enzyme “McsB” of B. subtilis was found to be a Tyr kinase, its Bacillus stearothermophilus homologue is hypothesized to be an Arg and not Tyr kinase [23, 24] Another unique class of enzymes possess both STPKs and Tyr kinase activities and are recognized as “dual specificity protein kinases” (DSPKs) [25, 26]. Both the novel kinases (Bas2152 and Bas2037) and the lone Ser/Thr protein phosphatase PrpC (BA-Stp1) were found to have overlapping specificities toward Tyr. Detailed experimental analysis showed that both kinases belong to separate classes of dual specificity protein kinases, with PrkD being the first bacterial “dual specificity tyrosine phosphorylation-regulated kinase” (DYRK). Pyruvate kinase (BasPyk) was identified as a substrate of PrkD, which was phosphorylated on Ser/Thr residues but not on Tyr, affirming that PrkD is a DYRK-like kinase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION