Interdomain Interaction Reconstitutes the Functionality of PknA, a Eukaryotic Type Ser/Thr Kinase from Mycobacterium tuberculosis

Abstract

Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality.

A Conserved Threonine Residue in the Juxtamembrane Domain of the XA21 Pattern Recognition Receptor Is Critical for Kinase Autophosphorylation and XA21-mediated Immunity

“…These results suggest that the amino acids immediately flanking Thr 705 are required for the residue to function. This is consistent with previous reports that the flanking sequence is required for kinase autophosphorylation (41,42).…”

Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis*

“…D, trans-phosphorylation activity of KD and full-length ICD constructs was compared using GarA as a substrate. The autoradiogram confirms that the PknA ICD is more active than the PknA KD as reported previously (38), but that activity does not differ for the two PknB and PknL constructs. External mass calibration was performed prior to analysis using the standard LTQ calibration mixture.…”

“…It must be stressed that other factors, such as molecular scaffolding and co-expression, may influence the cross-phosphorylation patterns in vivo. Moreover, the constructs used for in vitro biochemistry lacked possible regulatory sites in the juxtamembrane segments (24,38), potentially masking additional functional relationships. In addition, the network architecture may be modified by the temporally restricted activity of the STPKs mediated, in part by localization or regulation of the antagonistic phosphatase (50).…”

“…We focused on the minimal KD constructs, because these segments contain conserved activation loop Thr residues that must be phosphorylated to elicit activity. Although the STPKs also contain juxta-membrane phosphorylation sites implicated in regulation or binding accessory proteins (38,39), phosphorylation of the activation loop Thr is the primary mechanism of activation. Moreover, the in vitro specificity of a number of KDs has been shown to recapitulate functional substrate phosphorylations in vivo (40).…”

Protein Kinase A (PknA) of Mycobacterium tuberculosis Is Independently Activated and Is Critical for Growth in Vitro and Survival of the Pathogen in the Host

“…6A). In vitro kinase assays have shown that the intracellular region consisting of the kinase and juxtamembrane domains is catalytically active (42). To delineate the domains of PknA required for cell survival in mycobacteria, we generated deletion mutants PknA KD (aa 1-271), PknA JM (aa 1-341), and PknA TM (aa 1-361) in pNit and pCiT vectors (Fig.…”