GpsB coordinates StkP signalling as a PASTA kinase adaptor in Streptococcus pneumoniae cell division

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure that affects StkP-dependent phosphorylation in vivo. Recent phosphoproteomic analyses revealed phosphorylation of GpsB at multiple Ser and Thr residues. Here, we confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. StkP has intrinsic kinase activity, but GpsB directly stimulates the autophosphorylation of StkP and phosphorylation of StkP substrates. In vitro, GpsB phosphoablative substitutions had a reduced potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, negatively affected GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. Bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that phosphorylation and dephosphorylation of GpsB dynamically modulate StkP activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring proper functioning of the StkP signaling pathway.