Division of Microbiology and Academy of Scientific and Innovative Research, CSIR, Central Drug Research Institute, Lucknow, 226031, India.
Appl Microbiol Biotechnol. 2014 Dec;98(24):10065-76. doi: 10.1007/s00253-014-5859-4. Epub 2014 Jun 17.
PknJ (Rv2088) is a serine/threonine protein kinase of mycobacteria which is present in Mycobacterium tuberculosis (MTB), but its gene is absent in Mycobacterium smegmatis (MS); a fast grower and nonpathogenic species of mycobacteria. The heterologous expression of MTB-specific PknJ in MS altered the growth of recombinant mycobacteria highlighting one of the characteristics of this protein. This nature of the protein was further confirmed when Mycobacterium bovis BCG (BCG) containing antisense copy of pknJ resulted in the increased growth of BCG. The real-time RNA quantification analysis pointed out toward increased expression of this protein during infection of THP-1 macrophage cells which further emphasized that the protein is essential for the intracellular survival of mycobacteria. The differential in gel electrophoresis (DIGE) data followed by mass spectroscopy suggested that PknJ is involved in regulation of pyruvate kinase A (Rv1617). Since pyruvate kinase (PK) A is one of the key enzymes which controls glycolytic cycle in mycobacteria, we looked for its interaction with PknJ during extracellular and intracellular growth of mycobacteria. In order to identify the specific residue(s) involved in post-translational modification, the phospho-null mutants of PK were generated, and their substrate specificities in response to PknJ were assessed through kinase assay. The findings thus underlined that the PK activity is predominantly dependent on the threonine residue at the 94(th) position and further suggested that this site may be plausible in intracellular survival of mycobacteria upon phosphorylation with PknJ.
PKnJ（Rv2088）是分枝杆菌中的丝氨酸/苏氨酸蛋白激酶，存在于结核分枝杆菌（MTB）中，但在非致病性快速生长分枝杆菌耻垢分枝杆菌（MS）中其基因缺失。在 MS 中异源表达 MTB 特异性 PknJ 改变了重组分枝杆菌的生长，突出了该蛋白的一个特征。当含有 pknJ 反义拷贝的牛分枝杆菌卡介苗（BCG）导致 BCG 生长增加时，进一步证实了该蛋白的这种性质。实时 RNA 定量分析指出，该蛋白在 THP-1 巨噬细胞感染期间表达增加，这进一步强调了该蛋白对于分枝杆菌在细胞内的生存是必需的。差异凝胶电泳（DIGE）数据随后进行质谱分析表明，PKnJ 参与调节丙酮酸激酶 A（Rv1617）。由于丙酮酸激酶（PK）A 是控制分枝杆菌糖酵解循环的关键酶之一，我们在分枝杆菌的细胞外和细胞内生长过程中寻找其与 PknJ 的相互作用。为了确定参与翻译后修饰的特定残基，生成了 PK 的磷酸化缺失突变体，并通过激酶测定评估了它们对 PknJ 的底物特异性。研究结果强调了 PK 活性主要依赖于 94（th）位的苏氨酸残基，并进一步表明该位点在 PknJ 磷酸化后可能在分枝杆菌的细胞内生存中具有合理性。
