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Phosphorylation of mycobacterial phosphodiesterase by eukaryotic-type Ser/Thr kinase controls its two distinct and mutually exclusive functionalities

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Phosphorylation of mycobacterial phosphodiesterase by eukaryotic-type Ser/Thr kinase controls its two distinct and mutually exclusive functionalities
 
Creator Malhotra, Neha
Karthikeyan, Subramanian
Chakraborti, Pradip K.
 
Subject QR Microbiology
 
Description Phosphorylation-mediated negative feedback regulation of cAMP levels by phosphodiesterase is well-established in eukaryotic cells. However, such a mechanism remains unexplored in prokaryotes. We report here the involvement of eukaryotic-type Ser/Thr kinases, particularly PknA in trans-phosphorylating phosphodiesterase from Mycobacterium tuberculosis (mPDE), that resulted in decreased enzyme turnover rate compared with its unphosphorylated counterpart. To elucidate the role of mPDE phosphorylation in hydrolyzing cellular cAMP, we utilized a phosphodiesterase knock-out Escherichia coli strain, ΔcpdA, where interference of endogenous eukaryotic-type Ser/Thr kinases could be excluded. Interestingly, the mPDE-complemented ΔcpdA strain showed enhanced cAMP levels in the presence of PknA, and this effect was antagonized by PknA-K42N, a kinase-dead variant. Structural analysis of mPDE revealed that four Ser/Thr residues (Ser-20, Thr-22, Thr-182, and Thr-240) were close to the active site, indicating their possible role in phosphorylation-mediated alteration in enzymatic activity. Mutation of these residues one at a time to alanine or a combination of all four (mPDE-4A) affected catalytic activity of mPDE. Moreover, mPDE-4A protein in kinase assays exhibited reduction in its phosphorylation compared with mPDE. In consonance, phosphoproteins obtained after co-expression of PknA with mPDE/S20A/T240A/4A displayed decreased phospho-signal intensities in immunoblotting with anti-phosphoserine/phosphothreonine antibodies. Furthermore, unlike mPDE, phospho-ablated mPDE-T309A protein exhibited impaired cell wall localization in Mycobacterium smegmatis, whereas mPDE-4A behaved similarly as wild type. Taken together, our findings establish mutually exclusive dual functionality of mPDE upon PknA-mediated phosphorylation, where Ser-20/Thr-240 influence enzyme activity and Thr-309 endorses its cell wall localization.
 
Publisher American Society for Biochemistry and Molecular Biology
 
Date 2017
 
Type Article
PeerReviewed
 
Relation http://dx.doi.org/10.1074/jbc.M117.784124
http://crdd.osdd.net/open/2054/
 
Identifier Malhotra, Neha and Karthikeyan, Subramanian and Chakraborti, Pradip K. (2017) Phosphorylation of mycobacterial phosphodiesterase by eukaryotic-type Ser/Thr kinase controls its two distinct and mutually exclusive functionalities. Journal of Biological Chemistry, 292 (42). pp. 17362-17374. ISSN 0021-9258