Protein Phosphatase 2A Inhibitors, I1PP2A and I2PP2A, Associate with and Modify the Substrate Specificity of Protein Phosphatase 1

Abstract

Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A.