Fig. 5.
CinF is a protein phosphatase that targets IκBα. (A) CinF does not detectably dephosphorylate FBP. FBP dephosphorylation was measured with recombinant CinF, ST0318, or its enzymatically inactive mutant ST0318Y347T. The reaction measures fructose-6-phosphate formation by coupling the reaction with exogenous phosphoglucose isomerase and glucose-6-phosphate dehydrogenase that reduces NADP+ to NADPH. The values of Km and Kcat were obtained by plotting the plot Lineweaver–Burk equation. (B) CinF and ST0318 are unable to hydrolyze the alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP). Testing proteins were individually assayed for their ability to remove phosphate from pNPP, and the amount of free phosphate was determined by the malachite green assay. The established protein phosphatase WipA and its enzymatically inactive mutant were included as controls. (C) ST0318 cannot inhibit NF-κB activation. HEK293T cells transfected with a NF-κB reporter plasmid and constructs that express ST0318 or CinF are shown. Luciferase activity indicative of NF-κB activation was measured after PMA induction. The expression of the relevant proteins was probed by immunoblotting with a Flag-specific antibody (Lower). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Data shown were one of three independent experiments done in triplicate with similar results. (D and E) Dephosphorylation of IκBα by CinF. Recombinant CinF was incubated in reactions that contain p-IκBα or p-IKKβ for 60 min. The phosphorylation status of these proteins was detected by immunoblotting with phospho-specific antibodies. The amounts of IκBα, IKKβ, and CinF in the reactions were determined by immunoblotting with the indicated antibodies, respectively (D). Dose-dependent activity of CinF toward p-IκBα. The indicated amounts of CinF or its CinFY362A mutant were incubated with aliquoted p-IκBα and dephosphorylation was evaluated by a phospho-specific antibody are shown. Note that CinFY362A had no impact on p-IκBα (Upper). Reaction time-dependent activity of CinF was measured by incubating 40 ng CinF with equal amounts of p-IκBα, and the dephosphorylation activity was similarly determined. Note that a similar amount of ST0318 did not detectably dephosphorylate p-IκBα even after extended incubation (E, Lower). (F) CinF does not remove phosphate from p-IκBβ. The indicated amounts of CinF were incubated with aliquots of p-IκBβ, and its phosphorylation status was determined by immunoblotting. A fraction of the reactions was probed for IκBβ and CinF, respectively.