FIGURE 4.

STEP dephosphorylates Pyk2 at Tyr⁴⁰². A, Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate (Perv) (1 mm). Mg-ATP, STEP (10 μm), and pervanadate (1 mm) were added as indicated. The Tyr(P)⁴⁰² signal was normalized to total Pyk2 levels. Error bars indicate the standard error of the mean (SEM) of at least three independent experiments (in this and subsequent panels). Asterisks indicate statistical significance as compared with control (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4). B, total brain lysates were heated at 65 °C for 20 min to inactive endogenous kinases and phosphatases. Treated lysates (50 μg) were incubated with purified GST-STEP₄₆ WT (active) or GST-STEP₄₆ C/S (inactive) at the indicated concentrations and blotted with Tyr(P)⁴⁰² Pyk2 or Tyr(P)¹⁴⁷² NR2B. Quantitative analyses for each were normalized to GAPDH (*, p < 0.05; **, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 3). C, full-length pcDNA3-Pyk2 (1 μg) construct was co-transfected with pcDNA3-STEP₆₁ WT (active) or pcDNA3-STEP₆₁ C472S (inactive) construct into HEK293 cells. Thirty-six hours post-transfection, cells were lysed in radioimmune precipitation assay buffer and blotted with antibodies as indicated in the figure.