Polycystin-1 inhibits eIF2α phosphorylation and cell apoptosis through a PKR-eIF2α pathway

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 which encodes polycystin-1 (PC1) and polycystin-2, respectively. PC1 was previously shown to slow cell proliferation and inhibit apoptosis but the underlying mechanisms remain elusive or controversial. Here we showed in cultured mammalian cells and Pkd1 knockout mouse kidney epithelial cells that PC1 and its truncation mutant comprising the last five transmembrane segments and the intracellular C-terminus (PC1-5TMC) down-regulate the phosphorylation of protein kinase R (PKR) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). PKR is known to be activated by interferons and dsRNAs, inhibits protein synthesis and induces apoptosis. By co-immunoprecipitation experiments we found that PC1 truncation mutants associate with PKR, or with PKR and its activator PACT. Further experiments showed that PC1 and PC1-5TMC reduce phosphorylation of eIF2α through inhibiting PKR phosphorylation. Our TUNEL experiments using tunicamycin, an apoptosis inducer, and GADD34, an inhibitor of eIF2α phosphorylation, demonstrated that PC1-5TMC inhibits apoptosis of HEK293T cells in a PKR-eIF2α-dependent manner, with concurrent up- and down-regulation of Bcl-2 and Bax, respectively, revealed by Western blotting. Involvement of PC1-regulated eIF2α phosphorylation and a PKR-eIF2α pathway in cell apoptosis may be an important part of the mechanism underlying ADPKD pathogenesis.

Role of PKR in the Inhibition of Proliferation and Translation by Polycystin-1

“…Furthermore, it was found that mTOR and PKR may regulate the expression of PP2A subunit B56 α independently of their kinase activity [39], while results from our current experiments showed that siRNA of B56 α also abolishes PC1-inhibited proliferation and translation (data not shown). Therefore, we carried out co-IP experiments to document the physical interaction of PC1 with mTOR and PKR, and found that PC1-5TMC is in the same complex with mTOR and PKR in HeLa cells (Figures 4(a) and 4(b)), which is in line with previous reports [33, 40]. The results suggested that the PC1-PKR-mTOR association may mediate the effect of PC1 on proliferation and translation.…”

“…HeLa cells overexpressing eIF2 α exhibited much reduced proliferation rates, as expected, and were still inhibitable by PC1-5TMC (Figure 2(a)), indicating that the eIF2 α activity and PC1 inhibit proliferation through two different pathways. In fact, if inhibition by PC1 were through eIF2 α , then because PC1-5TMC reduces the eIF2 α activity [33], a known proliferation and translation inhibitor, we would see a stimulating effect of PC1-5TMC on proliferation, against our observation (Figure 2(a)). PC1-5TMC also inhibited protein synthesis assessed by 35 S labelling, but because overexpressed PKR almost completely stopped 35 S labelling, the effect of coexpressed PC1-5TMC on protein synthesis cannot be evaluated (Figure 2(b)).…”

“…We found that PC1 reduces apoptosis by inhibiting the PKR kinase activity and the phosphorylation of eIF2 α [33]. Here we tested whether PKR is involved in PC1-inhibited proliferation.…”

“…In particular, PKR as a kinase phosphorylates downstream substrates through which it regulates proliferation, translation, and apoptosis [28, 29]. In our previous study, we found that PC1 and PC1 truncate mutation inhibit P-eIF2 α through reducing kinase activity of PKR [33]. Therefore, it is likely that the PC1-inhibited cell proliferation should be through a signaling that is different from PKR-eIF2 α pathway, because suppressed PKR-eIF2 α activity would promote cell proliferation and protein translation.…”

“…Plasmid pcDNA3-GFP-PC1-5TMC(PC1-5TMC, aa 3895-4302) comprising last 5 TMs plus C-terminus was constructed using Stratagene Quik Change® II XL Site-Directed Mutagenesis Kit (Agilent Technologies Canada Inc., Mississauga, ON) as described previously [33]. Plasmid eIF2 α was from Santa Cruz (Santa Cruz, CA).…”