In vitro phosphorylation of membrane-associated protein fraction and mtPykA by PknJ-KD.

(A) 20 µg purified fraction of membrane-associated proteins was incubated with 2 µg of PknJ-KD or PknJ-KD-K43A, in an in vitro kinase assay. Samples were separated by 10% SDS-PAGE and autoradiographed on PhosphorImager. (B) Graph showing time-dependent phosphorylation of mtPykA by PknJ-KD. 1 µg of PknJ-KD was incubated with 2 µg of mtPykA with increasing time-points (0–30minutes) in in vitro kinase assay. Phosphorylation was evaluated as before. Experiment was performed twice and the results indicate average of the two. (C) Phosphoamino acid content of mtPykA phosphorylated by PknJ-KD was assessed as discussed earlier. (D) Time-dependent dephosphorylation of in vitro phosphorylated PknJ-KD (left panel) and mtPykA (right panel) by Mstp_cat. Extent of dephosphorylation was measured by adding 500 ng of purified Mstp_cat to a reaction mixture containing phosphorylated PknJ-KD and mtPykA for increasing time-points. Dephosphorylation was evaluated as discussed before with signal intensity at 0 minute taken as maximum. Experiment was performed twice and the results indicate average of the two.