Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the α subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2α in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr451) and eIF2α (Ser51) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2α phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2-). An inhibitor of p38, SB 202190 or a DNM of p38α kinase (DNM-p38α) suppressed UVA-induced phosphorylation of eIF2α (Ser51) but had no effect on phosphorylation of PKR (Thr451). Our data indicated that phosphorylation of PKR at Thr451 is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser51 phosphorylation of eIF2α in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2α at Ser51 occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.
Bibliographical noteFunding Information:
We thank Dr Takayasu Date for the generous gift of plasmids pEGST-PKR and pGEX-eIF2α, Dr John C.Bell for the generous gift of PKR+/+ and PKR−/− cell lines and identification of these cells and Dr Scot Kimball for the gift of non-phospho-eIF2α antibody. We thank Ms Andria Hansen for her secretarial assistance. This work was supported in part by The Hormel Foundation and National Institutes of Health grants CA77646, CA111356 and CA111536. The University of Minnesota is an equal opportunity educator and employer.