Figure 5. Factors affecting PstP activity.

(A) Auto-dephosphorylation of PstP_c: Autoradiogram showing phosphorylation by PknB_c. PstP_c and PstP_c ^D38G (3 µg each) were used for in vitro phosphorylation assay by PknB_c and PknB_c ^T171/173D (2 µg each). Since PknB_c ^T171/173D cannot be dephosphorylated by PstP_c, lack of signal signifies auto-dephosphorylation of phosphatase. PstP_c ^D38G was used as positive control to show that PknB_c ^T171/173D is active. Regulation of PstP_c activity: pNPP assay showing the effect on activity of PstP_c (1 µg) by (B) Zn²⁺ and (C) Pi. pNPP assay was carried out for 30 mins and activity was calculated as a measure of µmoles of pNPP hydrolyzed per min per µg of protein. The error bars show SD of three independent experiments. (D) Phosphorylation of PstP_c: Autoradiogram showing the phosphorylation of PstP_c (1 µg) by GST-PknA_c (left panel) and GST-PknB_c (right panel) in presence of 0.2 mM Zn²⁺ and 0.5 mM Pi. Since His₆-tagged STPKs were not resolved properly from PstP_c on SDS-PAGE (Figure S5), the assay was also performed with GST-tagged kinases having higher molecular weights. (E) Metabolic labeling of PstP_c by PknA and PknB in E. coli in presence of Zn²⁺ and Pi: Phosphorylation level of PstP_c was observed to be increased when Zn²⁺ (4 mM) and Pi (2 mM) were added during the culture conditions and subsequent processing steps. The autoradiograms obtained after SDS-PAGE were analyzed by ImageGauge software and intensity of the band corresponding to PstP_c phosphorylation without any added factor was taken as 100%. Relative phosphorylation is depicted in the bar graph.