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Molecular and Cellular Biology, March 2001, p. 1908-1920, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1908-1920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

Gregory A. Peters,1,2 Rune Hartmann,1,dagger Jun Qin,3 and Ganes C. Sen1,2,*

Department of Molecular Biology1 and Department of Molecular Cardiology,3 Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, and Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 441062

Received 3 August 2000/Returned for modification 11 September 2000/Accepted 6 December 2000

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.

* Corresponding author. Mailing address: Department of Molecular Biology/NC20, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Phone: (216) 444-0636. Fax: (216) 444-0513. E-mail: seng{at}ccf.org.

dagger Present address: Aarhus University, Aarhus, Denmark.

Molecular and Cellular Biology, March 2001, p. 1908-1920, Vol. 21, No. 6
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.6.1908-1920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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