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

Abstract

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/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 s20,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 a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Amplification of dopaminergic signaling by a positive feedback loop

“…Usui et al reported that the regulatory BЈ␦ subunit (or B56␦), originally purified from erythrocytes and named the BЉ (␦) subunit, is phosphorylated by PKA and that phosphorylated forms of BЈ␦ stimulate the catalytic activity of PP-2A in a substrate-specific manner (22,23). Immunoblot analysis using BЈ␦ antibody revealed that the level of expression of the protein was slightly higher in striatum than in neocortex (A.N., unpublished observations), the region in the central nervous system in which the level of expression of BЈ␦ was reported to be most abundant (24). BЈ␦ contains two consensus phosphorylation site motifs for PKA, and one or the other of these sites is also conserved in other B-type subunits, for example in B56␥3 (25).…”

Reduced expression of the A? subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the A? and A? subunit genes

“…22 Importantly, we have shown that in every case the mutation renders the A␣ subunit defective in binding B or C subunits, depending on whether the mutation is located in the N-terminal B subunit binding domain (repeats 1-10) or in the C-terminal C subunit binding region (repeats [11][12][13][14][15] (Fig. 1).…”

“…The A and C subunits both exist as 2 isoforms (␣ and ␤ ), whereas the B subunits fall into 3 families designated B, [2][3][4][5] , BЈ (also called B56) 6 -10 and BЉ. [11][12][13][14] A model demonstrating how the A, B and C subunits interact in the holoenzyme is shown in Figure 1. 15,16 The existence of so many regulatory subunits suggests that the various forms of PP2A have distinct functions.…”

Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the Aβ subunit gene

“…The B' family consists of numerous isoforms and splice variants whose molecular masses range from 54 to 68 kDa (Csortos et al, 1996;McCright et al, 1996;McCright and Virshup, 1995;Tanabe et al, 1996;Tehrani et al, 1996). The B'' family has at least four members, designated PR48 (Yan et al, 2000), PR59 (Voorhoeve et al, 1999), and PR72/PR130 (Hendrix et al, 1993;Nagase et al, 1997). PR72 and PR130 are splice variants of the same gene (Hendrix et al, 1993).…”