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

Abstract

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p-T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant-negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time-dependent augmentation of AKT S473 and GSK-3alpha S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK-3alpha was not dependent on PI3K activity. PKR inhibition augmented levels of p-S473 AKT and p-S21/9 GSK-3alpha/beta in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK-3alpha or beta phosphorylation in the presence of the AKT inhibitor, A443654. Pre-treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2alpha, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2alpha phosphorylation. The effects of PKR inhibition on AKT and GSK-3 phosphorylation were found to be, in part, PP2A-dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK-3.

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

“…As all previous reports of nuclear p-T451 PKR employed only immunocytochemical or basic western blotting techniques, we sought to characterize nuclear PKR in acute leukemia cell lines in which we demonstrated a requirement for PKR activity for proliferation and survival. 8 Using both proteomic and biochemical approaches, we demonstrate that there are diverse molecular weight and post-translationally modified forms of PKR in the nuclear compartment of acute leukemia cells, and that nuclear PKR is an active kinase which responds to stress. These data add significance to the clinical findings of p-T451 PKR in the nucleus and suggest a more direct role for PKR in nuclear signaling.…”

“…8 Primary antibodies were as follows (from Cell Signaling Laboratories, Danvers, MA, USA: a-p-T451 PKR (1:1000); Santa Cruz Biotechnologies, La Jolla, CA, USA: a-PKR (D-20; 1:1000), a-PKR (K-17; 1:1000), a-eIF2a (FL-315; 1:1000) and a-lamin B (1:1000); Abcam, Cambridge, UK: a-N-terminal PKR (ab58301; 1:500) and a-C-terminal PKR (ab28943; 1:500) or Sigma-Aldrich, St Louis, MO, USA: a-p-T451 PKR (1:1000) and a-b-tubulin (1:5000)). Secondary antibodies (Santa Cruz Biotechnologies) were diluted 1:10 000 in 3% BSA in PBST or 1:2000 in nonfat dry milk in PBST (p-T451 PKR).…”

“…Protein concentration was determined as described. 8 IPG gel strips (IPG ReadyStrip (pH 3-10) length 7 cm, Bio-Rad Laboratories Inc.,) were rehydrated in swelling solution (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM dithiothreitol and 0.2% anfoline) containing 500 mg of protein for 16 h at 20 1C. Isoelectric focusing was carried out for 30 min at 500 V (gradient), 60 min at 500 V (gradient), 60 min at 1000 V (gradient) and 4000 V to reach a total of 80 kVh.…”

“…7 In contrast, enhanced PKR activity has been reported in acute leukemia, colon, breast, lung and prostate cancer cell lines, which are deficient in PTEN, as well as patient samples from breast carcinoma, colon cancer, melanoma and diverse hematological malignancies. [8][9][10][11][12][13] Cancers possessing enhanced PKR activity are highly susceptible to inhibition of PKR. 8,13,14 Although PKR has been identified in nuclear and cytoplasmic fractions, most activities attributed to PKR occur in the cytoplasm; hence, the role of nuclear PKR has remained unclear.…”

“…[8][9][10][11][12][13] Cancers possessing enhanced PKR activity are highly susceptible to inhibition of PKR. 8,13,14 Although PKR has been identified in nuclear and cytoplasmic fractions, most activities attributed to PKR occur in the cytoplasm; hence, the role of nuclear PKR has remained unclear. Jeffrey et al 15 found, in NIH3T3 cells overexpressing exogenous PKR and Daudi Burkitt's lymphoma cells treated with interferon, the ratio of cytoplasmic to nuclear PKR to be 5:1.…”