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

Author:

Peters Gregory A.12,Hartmann Rune1,Qin Jun3,Sen Ganes C.12

Affiliation:

1. Department of Molecular Biology 1 and

2. Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 441062

3. Department of Molecular Cardiology, 3 Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, and

Abstract

ABSTRACT 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.

Publisher

American Society for Microbiology

Subject

Cell Biology,Molecular Biology

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