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

Faculty of Health Sciences and the Department of Pathology and Molecular Medicine, McMaster University, and the Father Sean O'Sullivan Research Centre, St. Joseph's Healthcare, Hamilton, Ontario, CANADA

^* To whom correspondence should be addressed. Email: mahonyj{at}mcmaster.ca.

	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 eukaryotic-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 E. 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-S-transferase fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated and catalytic mutants (K33G, D156G, and [K33G; D156G]) 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 the first to show phosphorylation of a predicted type III secretion structural protein.