Figure 2.
TbPIP39 is a phosphatase with regulatory interaction with TbPTP1. (A). Recombinant TbPIP39 was incubated with FGR kinase to generate a tyrosine-phosphorylated form over the course of 120 min. Tyrosine phosphorylation of the recombinant protein was detected using the phophotyrosine-specific antibody 4G10 (α-pTyr; Millipore). The right panel shows the same analysis using a mutant form of TbPIP39 in which the predicted tyrosine phosphorylation site Y278 is mutated to phenylalanine (F278). In this case, α-pTyr reactivity is lost. (B) Tyrosine-phosphorylated TbPIP39 is a substrate of TbPTP1. TbPIP39 was incubated with FGR kinase to generate tyrosine-phosphorylated protein, this being detected with the 4G10 antibody (α-pTyr). Upon incubation with wild-type TbPTP1, tyrosine phosphorylation of TbPIP39 is lost. In contrast, when incubated with either buffer alone or a catalytically dead (C229S) TbPTP1 mutant, TbPIP39 remains phosphorylated. (C) Activity against pNPP of recombinant TbPIP39 in the absence (column 1) or presence (column 2) of Mg2+. Column 3 shows the same assay as in column 2, using a dEdE mutant of TbPIP39. Columns 4–7 show the activity against pNPP of TbPTP1 alone (column 4) or in the presence of 1 μg (column 5), 0.1 μg (column 6), or 0.01 μg of TbPIP39. No Mg2+ was included in the reactions in columns 4–7, preventing TbPIP39 activity. Statistical analyses used a general linear model. (*) P < 0.05; (**) P < 0.001. (D) Activity against pNPP of recombinant TbPIP39 as it becomes tyrosine-phosphorylated by FGR kinase. With increasing phosphorylation, the activity increases. (Right graphs) In TbPIP39-Y278F, no enhanced activity is observed, demonstrating that tyrosine phosphorylation on Y278 is responsible for TbPIP39 regulation. The input recombinant proteins are those depicted in A. Statistical analyses used a general linear model. (*) P < 0.05.