One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR). PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein-protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and inter action with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that, although dispensable for PACT's high-affinity interaction with PKR, the third motif is essential for PKR activation. In addition, domain 3 and eukaryotic initiation factor 2alpha both interact with PKR through the same region within PKR, which we have mapped to lie between amino acid residues 318 and 551.

Phyllodes tumors (PTs) of the breast are biphasic lesions, comprising an epithelial component set within a neoplastic spindle-celled stroma. These tumors have been classified as benign, borderline, and malignant based on a combination of histological criteria, including stromal cellularity, nuclear pleomorphism, mitotic rate, stromal overgrowth, and margin appearance. The aim of this study was to evaluate the expression of CD34, CD117 (c-kit), and Ki-67 in PT of the breast and attempt to correlate the staining pattern with tumor grade by morphology. Immunohistochemical expression of CD117, CD34, and Ki-67 was studied on formalin-fixed, paraffin-embedded archival tissue material from 33 cases of PT. Histologically, there were 21 benign, 6 borderline, and 6 malignant (high-grade) tumors. All 6 histologically malignant PTs were positive for CD117 (100%), but only 1 marked with CD34 (16.7%). Borderline PTs frequently coexpressed CD34 and CD117 (66.7%). The benign PTs, on the other hand, most commonly (52.4%) showed a CD34(+)/CD117(-) immunoprofile with 33.3% cases coexpressing the markers: that is, CD34(+)/CD117(+). Although most benign PTs (80.6%) showed a Ki-67 of <2%, a few cases showed slightly higher proliferation indices. This study indicates that CD34 and CD117 are differentially expressed in benign and malignant PTs. These markers, therefore, in combination, may be used as an adjunct to morphology in the subclassification of PTs.

PKR is an interferon‐induced serine‐threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR's kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double‐stranded (ds) RNA serves as PKR's activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP‐binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the α‐subunit of the protein synthesis initiation factor 2 (eIF2α) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA‐induced activation of PKR, heparin‐dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin‐induced PKR activation, we have mapped the heparin‐binding domains of PKR. Our results indicate that PKR has two heparin‐binding domains that are nonoverlapping with its dsRNA‐binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin‐binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.