PKR Promotes BCL2 Phosphatase Function in ALL Cells by Dual Mechanisms: As a B56α Kinase and by Supporting B56α Expression Via eIF2α And the PI3K/AKT Cascade.
Authors: Ruvolo Peter P. , Ruvolo Vivian R 
Published: April 2020

Abstract
Activation of double-stranded RNA-activated Protein Kinase (PKR) is associated with growth inhibition and cell death. PKR is normally dormant in cells and is activated in response to stress challenge (e.g. viral infection, chemotherapy, factor withdrawal). The presence of active kinase under growth conditions is generally not observed. PKR expression and activation status was determined in four ALL cell lines: REH, CCRF-CEM, MOLT4, and RS(4;11). PKR was found to be expressed and phosphorylated in all the cell lines. The presence of active PKR suggests the kinase may be important for cellular homeostasis in ALL cells. The best characterized substrate of PKR is eIF2α. Phosphorylation of eIF2α was examined to determine if PKR phosphorylation levels correlated with phosphorylation of the kinase’s primary substrate. Phosphorylated eIF2α was detected in all the cell lines except RS(4;11) cells. The lack of observed phosphorylated eIF2α in the RS(4;11) cells was due to the low abundance of the eIF2α protein. Another substrate of PKR is B56α, a B regulatory subunit of Protein Phosphatase 2A (PP2A). B56α comprises a mitochondrial PP2A isoform that serves as the BCL2 phosphatase and promotes cell death in response to chemotherapeutic agents. PKR was found to phosphorylate B56α at serine 28 in REH cells to promote mitochondrial PP2A activity and BCL2 phosphatase function. Loss of PKR by shRNA results in phosphorylation of BCL2 and the cells become resistant to the chemotherapeutic drug etoposide. Inhibition of PKR by pharmacologic inhibitor or shRNA in REH cells results in loss of B56α expression suggesting that phosphorylation may be required for stability of the B subunit. However, phosphorylation of B56α is not required for its stability since mutant S28A B56α protein is readily expressed in cells. A clue to an alternative mechanism was provided by the eIF2α phosphorylation status and B56α expression pattern in the four ALL cell lines. RS(4;11) cells display little if any B56α protein while the other three ALL cell lines express the B subunit. REH, CCRF-CEM, and MOLT4 cells exhibited ~ 3X higher mitochondrial PP2A activity compared to RS(4;11) cells. The lack of mitochondrial PP2A activity in RS(4;11) cells is likely due to a lack of B56α. Since RS(4;11) cells have active PKR but lack phosphorylated eIF2α, the possibility arises that B56α expression may depend on phosphorylated eIF2α. The mechanism how PKR promotes B56α expression appears to involve the proteasome since a proteasome inhibitor can block loss of the B subunit when PKR is suppressed. A role for eIF2α is suggested since salubrinal, a drug that promotes eIF2α phosphorylation by preventing the dephosphorylation of the molecule increased expression of B56α protein. It has recently been found that eIF2α phosphorylation can activate the PI3K/AKT signaling cascade. Suppression of PKR in REH cells blocks AKT phosphorylation suggesting that PKR may positively regulate AKT in these cells. It appears that the mechanism how PKR promotes B56α expression involves the PI3K/AKT cascade since inhibition of this signaling pathway using LY294002 blocks expression of the B subunit. Since B56α mediated mitochondrial PP2A activity promotes cell death in response to chemotherapeutic agents, it is likely that the normally pro-survival PI3K/AKT pathway may serve a role in stress signaling in ALL cells by supporting B56α expression to promote BCL2 phosphatase activity. These findings indicate that PKR can regulate the BCL2 phosphatase at multiple levels in ALL cells. Understanding the complex regulatory pathway how PKR controls PP2A and BCL2 will be necessary for designing effective therapies for the treatment of ALL.