Fig. 6
Spr1851/Jag is a new substrate of StkP and PhpP. a Identification of Jag. Membrane fraction isolated and extracted by TFE/chloroform from strain Sp169 (ΔphpP ΔdivIVA) was resolved on 2D SDS-PAGE, immunobloted with α-pThr antibody and matched with parallel gel stained with Coomassie Blue. Spot corresponding to P40 was excised, analyzed by MALDI-MS and identified as Spr1851. b Schematic structure of Spr1851/Jag. Jag_N: conserved domain found at the N-terminus of Jag proteins; T89: phosphorylated threonine 89; KH: single-stranded RNA binding domain; R3H: putative single-stranded nucleic acid binding domain. c Deletion of jag. Whole cell lysates of WT (Sp1), Δjag (Sp295), ΔphpP ΔdivIVA (Sp169) and ΔstkP (Sp10) were separated by SDS-PAGE and immunoblotted with α-pThr antibody to detect protein phosphorylation. Immunodetection of α-subunit of RNA polymerase (α-RpoA) was used as a loading control. Arrows indicate position of StkP and its substrates. d Phosphorylation of T89. Total protein lysates (30 μg) of WT (Sp1), Δjag (Sp295), complementation strain Δjag PZn-jag-flag (Sp304) and Δjag PZn-jag-flag-T89A (Sp302) were separated by SDS-PAGE and immunoblotted with α-pThr antibody to detect protein phosphorylation. Expression of Jag-Flag was monitored by immunodetection with anti-Flag antibody (α-Flag) and immunodetection of RpoA (α-RpoA) was used as a loading control. Arrows indicate position of proteins. e Dephosphorylation of Jag. Purified Jag-Flag was incubated with His-PhpP in vitro and reaction was stopped at given time. Samples were subjected to SDS-PAGE and immunoblotted with α-pThr antibody to visualize dephosphorylation in time. PhpP-His was detected with α-PhpP antibody and Jag-Flag was detected with α-Flag antibody. To exclude the spontaneous decay, phosphorylated form of Jag-Flag was incubated for 90 min in phosphatase reaction buffer without addition of His-PhpP. Arrows indicate position of two differentially phosphorylated forms of Jag