Tissue and Subcellular Distributions, and Characterization of Rat Brain Protein Phosphatase 2A Containing a 72-kDa /B'' Subunit

Abstract

A 74-kDa δ/B″ subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2 A (PP2A) consisting of a 34-kDa catalytic subunit (α/C) and 63- and 74-kDa regulatory subunits (β/A and δ/B″) in a ratio of 1:1:1. The purified δ/B″ was used as an immunogen in mice, to prepare specific antisera against δ/B″. Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s_{20, w} value of 7.2 ±0.3 S and the Stokes radius of 5.5 ±0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC₅₀ for okadaic acid of 10⁻⁹ M; (2) its preferential dephosphorylation of the α subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The K_m value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 ± 0.3 μM. and 192±2mol P_i released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (α₁β₁/ CAB″). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.