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

Vivian R Ruvolo,Svitlana M Kurinna,Kul B Karanjeet,Todd F Schuster,Alberto M Martelli,James A Mccubrey,Peter P Ruvolo

doi:10.1074/jbc.m800951200

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 B56alpha is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56alpha at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56alpha 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 B56alpha-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56alpha 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 B56alpha proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56alpha protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2alpha activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56alpha, because wild-type but not mutant S28A B56alpha promoted mitochondrial PP2A activity. Cells expressing wild-type B56alpha but not S28A B56alpha were sensitized to etoposide. These results suggest that PKR regulates B56alpha-mediated PP2A signaling in REH cells.

Highlights

phosphatase 2A (PP2A) function is regulated both negatively and positively by post-translational modification (4, 16 –18)
We examined whether PKR is a physiologic B56␣ kinase in REH cells and how the kinase might regulate B56␣-mediated PP2A function in these cells
PKR Is Basally Active in a Number of acute lymphoblastic leukemia (ALL) Cell Lines Including REH—PKR is thought to be normally dormant in cells and is expected to be activated only in response to viral infection or stress challenges [22]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—PhoenixTM Ampho retroviral packaging cells were obtained from Orbigen (San Diego, CA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% newborn calf serum and 5% fetal bovine serum at 37 °C in 5% CO2. A plasmid from GeneCopoeia containing HA-tagged GFP under the cytomegalovirus promoter was used To obtain the latter we used a QuikChange௡ XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) as directed by the manufacturer. Retroviral Transduction of shRNA—HuSH shRNA (29-mer) targeting PKR and a negative control plasmid targeting GFP were purchased from OriGene (Rockville, MD) These were transiently transfected into PhoenixTM Ampho cells using LipofectamineTM 2000 (Invitrogen) as directed by the manufacturer. Protein Phosphatase Assay—Protein phosphatase activity of isolated mitochondria and nuclei were determined as described previously [12]. The phosphatase assay was performed in a PP2A-specific reaction buffer (50 mM imidazole, pH 7.2, 0.2 mM EGTA, 0.02% 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin) using 100 ␮M phosphopeptide substrate and 2 ␮g of protein mitochondria or nuclei isolated as described above.

RESULTS

If PKR regulates a mitochondrial

DISCUSSION