Modular structure of PACT: distinct domains for binding and activating PKR.
PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.
Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
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Peters
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Hartmann
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Sen
D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005347:Fibroblasts; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D019892:eIF-2 Kinase / Q000378:metabolism*
D002352:Carrier Proteins; C104647:PRKRA protein, human; C119610:Prkra protein, mouse; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D019892:eIF-2 Kinase; D015088:2',5'-Oligoadenylate Synthetase