Yersinia protein kinase A autophosphorylation: Characterization of site-specific mutations

Pathogenic Yersinia species use the type III secretion system to inject effector proteins into host cells.  One of these effectors, the Yersinia protein kinase YpkA, is an essential virulence protein involved in rearrangement of the host actin cytoskeleton and inhibition of phagocytosis.  In a mouse infection model, the kinase activity of YpkA was important for Yersinia virulence.  Upon translocation, the C-terminal domain of YpkA binds to actin and autophosphorylates on serine residues resulting in increased kinase activity.  It has been proposed that actin binding triggers autophosphorylation on Ser90 and Ser95 of YpkA in vitro.  Here, we report the identification of 15 additional potential autophosphorylation sites.  We generated recombinant GST-tagged YpkA serine to alanine mutants and performed in vitro kinase assays.  Our results revealed Ser144Ala, Ser317Ala, and Ser320Ala had decreased autophosphorylation and substrate phosphorylation when compared to wildtype.  We previously demonstrated that YpkA inhibits the host Gαq signaling pathway through phosphorylation of Gαq.  To gain a better understanding of the serine residues that are critical for YpkA kinase activation in vivo, we performed an immunofluorescence study using HEK cells and GFP-tagged Tubby, a putative transcription factor that translocates to the nucleus upon Gαq activation, as readout for Gαq activation.  We have shown that the YpkA mutants (YpkAS90A/S95A, YpkAS144A, YpkAS90A/S95A/S144A, and YpkAS320A) are able to inhibit Gaq signaling similar to wildtype YpkA, suggesting the presence of additional sites in vivo.  Identification of the critical autophosphorylation sites will lead to new insights on YpkA kinase activation and its role in Yersinia virulence.