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Evidence for the interplay between post‐translational modifications in regulating mycobacterial sirtuin activity
Author(s) -
Yadav Ghanshyam Singh,
Ravala Sandeep K,
Malhotra Neha,
Chakraborti Pradip K
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1131.1
Subject(s) - sirtuin , phosphorylation , biology , kinase , biochemistry , threonine , serine , protein phosphorylation , protein kinase a , acetylation , microbiology and biotechnology , nad+ kinase , enzyme , gene
Regulation of cellular processes by post‐translational modification of proteins is well known throughout the phylogeny. Phosphorylation/dephosphorylation of a given protein in bacteria is often controlled by Eukaryotic‐type serine/threonine kinases and cognate phosphatases. Similarly bacterial proteins are also governed by acetylation/deacetylation events. In this context, we focussed on only sirtuin, the NAD + ‐dependent deacetylase present in the Mycobacterium tuberculosis genome, involved in removal of acetyl group from Nɛ‐acetyl‐lysine of a protein. Sirtuin family of proteins are conserved throughout the phylogeny and in eukaryotes they are also regulated by different kinases affecting diverse cellular functions. This interplay of kinase(s) and sirtuins is not elucidated in prokaryotes. Utilizing in vitro and in vivo transphosphorylation assays we found PknA, an essential mycobacterial eukaryotic‐type serine/threonine kinase is able to phosphorylate only sirtuin (mDAC) of M. tuberculosis . Compared to unphosphorylated mDAC, phosphorylation of mDAC causes decrease in deacetylase activity. Utilizing mass spectrometry approach, we observed that different serine and threonine residues were phosphorylated. In vitro kinase assays with different phospho‐site mutants of mDAC identified Thr‐214 as the major phosphosite. Over‐expression of mDAC or T214A in Mycobacterium smegmatis , where endogenous kinases are present, yielded phosphorylated proteins. Expectedly, magnitude of phosphorylation, as assessed by monitoring band intensity in western blot using anti‐phosphothreonine antibody, was considerably low in T214A protein compared to that of the mDAC. We further noted that sirtuin knock‐out E. coli strain was compromised in its growth profile in nutrient deprived condition (acetate medium). This phenotype, however, was reversed upon mDAC expression. Interestingly, expression of PknA in the mDAC complemented E. coli strain exhibited growth inhibition in acetate medium, indicating phosphorylation mediated regulation of mycobacterial sirtuin. In consonance with this finding, a phosphomimic, T214E also exhibited growth compromised behaviour compared to that of the wild‐type. Our study therefore, draws its logical inspiration on the possibility of interactions of two distinct post‐translational events in successful pathogens like M. tuberculosis for their versatile metabolic adaptability. Support or Funding Information This study was supported by institutional (CSIR‐Institute of Microbial Technology, Chandigarh) research grants (OLP‐97) and also from Department of Science and Technology (JC Bose National Fellowship to PKC, No. SR/S2/JCB‐87/2012, Science and Engineering Research Board), New Delhi, India.