Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells.

Cell cycle checkpoints are surveillance mechanisms that monitor and coordinate the order and fidelity of cell cycle events. When defects in the division program of a cell are detected, checkpoints prevent the pursuant cell cycle transition through regulation of the relevant cyclin-cdk complex(es). Checkpoints that respond to DNA damage have been described for the G1, S and G2 phases of the cell cycle. The p53 tumour suppressor is a key regulator of G1/S checkpoints, and can promote cell cycle delay or apoptosis in response to DNA damage. The importance of these events to cellular physiology is highlighted by the fact that tumours, in which p53 is frequently mutated, have widespread defects in the G1/S DNA damage checkpoints and a heightened level of genomic instability. G2/M DNA damage checkpoints have been defined by yeast genetics, though the genes in this response are conserved in mammals. We show here using biochemical and physiological assays that p53 is dispensable for a DNA damage checkpoint activated in the G2 phase of the cell cycle. Moreover, upregulation of p53 through serine 20 phosphorylation, does not occur in G2. Conversely, we show that the Chk1 protein kinase is essential for the human G2 DNA damage checkpoint. Importantly, inhibition of Chk1 in p53 deficient cells greatly sensitizes them to radiation, validating the hypothesis of targeting Chk1 in rational drug design and development for anti-cancer therapies.

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-alpha.

We have examined the effects of human papilloma virus (HPV) E6 proteins on interferon (IFN) signaling. Here we show that expression of the 'malignant' HPV-18 E6 in human HT1080 cells results in inhibition of Jak-STAT activation in response to IFN-alpha but not IFN-gamma. This inhibitory effect is not shared by the 'benign' HPV-11 E6. The DNA-binding and transactivation capacities of the transcription factor ISGF3 are diminished in cells expressing HPV-18 E6 after IFN-alpha treatment as a result of decreased tyrosine phosphorylation of Tyk2, STAT2 and STAT1. However, HPV-18 E6 does not affect the induction of tyrosine phosphorylation and DNA-binding of STAT1 by IFN-gamma. In addition, HPV E6 proteins physically interact with Tyk2. This interaction takes place preferably with HPV-18 E6 and to a lesser extent with HPV-11 E6. The E6/Tyk2 interaction requires the JH6-JH7 domains of Tyk2, which are important for Tyk2 binding to the cytoplasmic portion of IFN-alpha receptor 1 (IFNAR1). These findings demonstrate an inhibitory role of HPV-18 E6 in the IFN-alpha-induced Jak-STAT pathway, which may be explained, at least in part, by the ability of E6 to interact with and impair Tyk2 activation.

Upregulation of STAT1 protein in cells lacking or expressing mutants of the double-stranded RNA-dependent protein kinase PKR.

The interferon (IFN)-inducible double-stranded (ds) RNA-dependent protein kinase PKR plays a role in the regulation of gene expression through its capacity to phosphorylate the translation initiation factor eIF-2 and to inhibit protein synthesis. In addition to translational control, PKR has been implicated in the regulation of gene expression at the transcriptional level. In this regard, we have reported that PKR participates in IFN-and dsRNA-mediated signaling pathways by interacting with and modulating the transcriptional activity of the signal transducer and activator of transcription STAT1 [Wong, A.H.-T., Tam, N.W.N., Yang, Y.-L., Cuddihy, A.R., Li, S., Kirchhoff, S., Hauser, H., Decker, T. & Koromilas, A.E. (1997) EMBO J. 16, 1291-1304]. Here we report that the STAT1 protein is upregulated in cells lacking PKR (PKR-/-) and in cells expressing dominant negative PKR mutants. This upregulation is specific for STAT1 as increased expression is not observed for other STAT proteins. The inhibitory effect of PKR on STAT1 expression is exerted at the post-translational level because PKR-/- cells exhibit higher STAT1 protein stability than PKR+/+ cells.

The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro.

The tumor suppressor p53 is a multifunctional protein that plays a critical role in modulating cellular responses upon DNA damage or other stresses. These functions of p53 are regulated both by protein-protein interactions and phosphorylation. The double-stranded RNA activated protein kinase PKR is a serine/threonine kinase that modulates protein synthesis through the phosphorylation of translation initiation factor eIF-2alpha. PKR is an interferon (IFN)-inducible protein that is thought to mediate the anti-viral and anti-proliferative effects of IFN via its capacity to inhibit protein synthesis. Here we report that PKR physically associates with p53. The interaction of PKR with p53 is enhanced by IFNs and upon conditions that p53 acquires a wild type conformation. PKR/p53 complex formation in vitro requires the N-terminal regulatory domain of PKR and the last 30 amino acids of the C-terminus of human p53. In addition, p53 may function as a substrate of PKR since phosphorylation of human p53 on serine392 is induced by activated PKR in vitro. These novel findings raise the possibility of a functional interaction between PKR and p53 in vivo, which may account, at least in part, for the ability of each protein to regulate gene expression at both the transcriptional and the translational levels.

Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53.

The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR+/+ and PKR-/- mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G0/G1 arrest were impaired in PKR-/- cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser18 was defective in PKR-/- cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21(WAF/Cip1) and Mdm2. In addition, Ser18 phosphorylation and transcriptional activation by mouse p53 were diminished in PKR-/- cells after DNA damage induced by the anticancer drug adriamycin or gamma radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser18 of p53 by adriamycin to a higher degree in PKR+/+ cells than in PKR-/- cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.

Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways.

The interferon-inducible double-stranded RNA protein kinase PKR controls protein synthesis through the phosphorylation of eukaryotic translation initiation factor (eIF)-2. In addition to its demonstrated role in translational control, several reports have suggested a transcriptional role for PKR. Here we report that PKR is involved in IFN- and dsRNA-signaling pathways by modulating the function of the signal transducer and activator of transcription STAT1. We also show that PKR associates with STAT1 in mouse and human cells. The association is not a kinase-substrate interaction since STAT1 phosphorylation is not modified by PKR in vitro or in vivo. In addition, the formation of the PKR-STAT1 complex is not dependent upon the enzymatic activity of PKR but does require the dsRNA-binding domain of PKR. Moreover, there is a concomitant decrease in PKR-STAT1 interaction and increase in STAT1 DNA binding in response to IFNs or dsRNA. These findings suggest that PKR plays an important role in IFN and dsRNA-signaling pathways by modulating the transcriptional function of STAT1.

Induction of CD4 expression and human immunodeficiency virus type 1 replication by mutants of the interferon-inducible protein kinase PKR.

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), and the IFN-inducible protein kinase PKR is thought to mediate this effect by regulating protein synthesis. Here we report that ectopic expression of dominant negative PKR mutants in Jurkat cells induces HIV-1 replication. Specifically, expression of CD4 is upregulated by the PKR mutants, and this correlates with an induction of HIV-1 binding and proviral DNA synthesis upon HIV-1 infection. Moreover, activation of NF-kappaB was induced by an RNA binding-defective mutant of PKR. Thus, it appears that PKR, in addition to translational control, is involved in HIV-1 replication by modulating virus binding through the regulation of CD4 expression and virus gene expression through the activation of NF-kappaB.