Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Abstract

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione Stransferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

Characterization of the Putative Type III Secretion ATPase CdsN (Cpn0707) ofChlamydophila pneumoniae

“…The absorbance was measured at 420 nm, and the ␤-galactosidase activity was expressed as units of ␤-galactosidase activity per milligram of bacteria. Empty pT18 and pT25 vectors were transformed into E. coli DHP1 cells as a negative control, and pT18-PknD and pT25-CdsD-FHA-2, which is the FHA-2 domain of CdsD, were used as a positive control (22). The cutoff for a positive interaction (677 units of activity/mg bacteria) was determined as the mean plus 2 standard deviations of the negative control values obtained from 20 assays.…”

CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity

“…While several bacterial PP2Cs have been shown to mediate P-Tyr dephosphorylation in vitro, the biological relevance of PP2C-mediated tyrosine dephosphorylation remains unknown, and the need for a tyrosine phosphatase in C. trachomatis is unclear (34,37,66). In Chlamydia, the Hanks'-type kinases PknD and Pkn1 have been functionally validated in C. trachomatis and C. pneumoniae (PknD only) (19,20). While PknD and Pkn1 from C. trachomatis were found to phosphorylate only Ser and Thr, the C. pneumoniae PknD could also phosphorylate Tyr.…”

“…Chlamydia spp. encode two validated (Pkn1 and PknD) and one putative (Pkn5) Hanks'-type kinases (19,20). Phosphoproteomic analysis of Chlamydia caviae identified 42 phosphoproteins that are present in a developmental stage-specific pattern, with EBs containing 3-fold more phosphoproteins than RBs (21).…”

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

“…The information acquired in the past few years point toward their roles in secretion pathways, transcription, cellular metabolism, and signal transduction (17)(18)(19)(20)(21). Among prokaryotes, Mycobacterium tuberculosis has emerged as an attractive model system for understanding FHA domain-mediated signaling for two reasons: 1) M. tuberculosis encodes for 11 eukaryotic like STPKs (PknA to PknL, except PknC) and seven FHA domains in six proteins (22), thus offering an experimentally malleable system to understand molecular themes common to prokaryotes and eukaryotes.…”