Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division.
A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.
Chaba
Rachna
R
Institute of Microbial Technology, Chandigarh, India.
Raje
Manoj
M
Chakraborti
Pradip K
PK
D001426:Bacterial Proteins; D002455:Cell Division; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000166:cytology* / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000145:classification* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C451020:PknA protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases