PKR Regulates B56α-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells

Abstract

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56␣ is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56␣ at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56␣ in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56␣-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56␣ phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56␣ proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56␣ protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2␣ activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56␣, because wild-type but not mutant S28A B56␣ promoted mitochondrial PP2A activity. Cells expressing wildtype B56␣ but not S28A B56␣ were sensitized to etoposide. These results suggest that PKR regulates B56␣-mediated PP2A signaling in REH cells.Protein phosphatase 2A (PP2A) 2 is an important regulator of apoptosis and may be a tumor suppressor (1-4). How PP2A might function as a tumor suppressor is not clear, as the enzyme on the one hand is required for cell survival but on the other is active in processes responsible for cell death (2-7). An explanation for this paradox is that PP2A is not a single enzyme but rather a family of protein phosphatase isoforms (3,4,8,9). PP2A is a heterotrimer composed of a catalytic (C) subunit, a scaffold (A) subunit, and a regulatory (B) subunit. The C and A subunits form a catalytic complex that interacts with one of at least 21 diverse B subunit members from three major families (i.e. B55, B56, and PR72/130; see Refs. 2, 3, and 9). It is becoming evident that the function of PP2A relies on the B subunit because the various B subunit proteins determine substrate specificity and PP2A complex subcellular localization (2, 3, 7, 10 -13). The recent crystal structure of PP2A supports this premise (4, 13-15). Thus the PP2A family of protein phosphatase isoforms can best be defined by its regulatory B subunit.PP2A function is regulated both negatively and positively by post-translational modification (4, 16 -18). Phosphorylation of B56␥ by ERK at serine 337 inhibits PP2A function (13, 18). Interestingly, this regulatory ERK site is conserved in all of the known B56 family members except B56␣. Although ERK may not regulate B56␣ function negatively, phosphory...

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PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

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