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

Abstract

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

Allelic methylation levels of the noncoding VTRNA2-1 located on chromosome 5q31.1 predict outcome in AML

“…19,44 In addition, novel studies show that PKR may have specific functions in the nucleus of leukemic blasts. 45 Although none of the reported functions of the mature miRs 41,46 seem to apply directly to the current study, this proposed negative regulatory function of vtRNA2-1 on PKR kinase activity, the increased chemosensitivity by PKR inhibition, and the need for For personal use only. on May 11, 2018.…”

The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G1arrest

“…However, while we could not detect nuclear RAX/PACT or PKR, others have reported that PKR may translocate into the nucleus during stress where a majority of Ubc9 and p53 are located. [44][45][46] Thus, it is possible that p53 sumoylation may occur in either the nucleus or cytoplasm or both locations, and the process may even be cell type-dependent. Further experiments will need to be performed to determine whether RAX/PACT-PKR transiently associates with p53 and Ubc9 only in the cytosol, or whether RAX/PACT-PKRdependent sumoylation and activation of p53 occurs following PKR translocation to the nucleus.…”

PKR inhibits the DNA damage response, and is associated with poor survival in AML and accelerated leukemia in NHD13 mice

“…16,17,20 To elucidate the role of PKR in leukemia, we tested whether PKR expression may be associated with clinical outcome by RPPA analysis of primary CD34 1 blast cells from 414 newly diagnosed AML patients. 22,23 When stratified for PKR, AML patients with the highest one-third of PKR expression in CD34 1 cells had a significantly worse overall survival (OS) than patients with the lowest two-third of PKR expression ( Figure 1A) (median survival 41.6 vs 54.6 weeks; P , .05).…”

“…17,19,20 Since we recently observed that Lineagenegative (Lin 2 ) cells isolated from BM of TgPKR mice are more sensitive, whereas cells from PKRKO mice are highly resistant to genotoxic stresses including ionizing irradiation (IR), 3 we investigated whether nuclear PKR may regulate DNA damage response (DDR) signaling and whether decreased PKR expression/activity may safeguard genomic fidelity. 1 cells was performed by reverse phase protein array (RPPA) using the PKR M02 monoclonal antibody, clone 1D11 (Abnova, Taipei, Taiwan).…”