Gregory A. Peters

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

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.

Inhibition of PACT-Mediated Activation of PKR by the Herpes Simplex Virus Type 1 Us11 Protein

ABSTRACT PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR.

Phosphorylation of specific serine residues in the PKR-activation domain of PACT is essential for its ability to mediate apoptosis

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.

The role of PACT in mediating gene induction, PKR activation, and apoptosis in response to diverse stimuli.

PACT, the protein activator of the double-stranded (ds)RNA-activated protein kinase (PKR) has been shown to strongly interact with and activate PKR in cultured cells and in vitro. To further analyze the functions of PACT we have recently generated PACT knockout (KO) mice and described several developmental defects that are absent in PKR KO mice. Importantly, PACT has been previously suggested to be involved in different signaling pathways that include endoplasmic reticulum stress, serum deprivation, growth factor withdrawal, viral infection, and cytokine responses. In this study, we have analyzed the contribution of PACT to these pathways using cells derived from wildtype (WT) and PACT KO mice. Notably, we have been unable to detect any significant differences in the responses to stress stimuli comparing WT and PACT KO cells, although we have been able to validate the specific interaction between PACT and PKR. Taken together, our results reinforce the importance of genetic loss of function analysis to infer protein function.

The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation

PACT is a double-stranded RNA-binding protein that also binds and activates the latent protein kinase, PKR, which plays a major role in cellular antiviral defense in mammals. For evaluating PACTs contribution to the innate immune system, Pact−/− mice have been generated; these mice exhibit notable developmental abnormalities including microtia, with craniofacial, ear, and hearing defects. Here we report that, in addition, Pact−/− mice had smaller body size and fertility defects, both of which were caused by defective pituitary functions. Pact−/− mice exhibited anterior pituitary lobe (AL) hypoplasia, which developed postnatally, when the second phase of pituitary expansion occurs. Among the 5 cell types in AL, the numbers of corticotrophs, gonadotrophs, and somatotrophs were equally decreased in Pact−/− mice with a greater impact on lactotrophs and a lesser impact on thyrotrophs. PACT mRNA and protein were highly expressed in the pituitary of wild-type (Wt) mice during the postnatal wave of AL proliferation, the same period in which the hypoplasia developed in Pact−/− mice. During this time, the pituitaries of Pact−/− mice did not exhibit significantly increased apoptosis compared with Wt mice but showed a decrease in cell proliferation. The inhibition of cell proliferation observed in vivo could be recapitulated in vitro in GH3 somato/lactotroph and LβT2 gonadotroph cell lines; knockdown of PACT expression with siRNA diminished the rate of proliferation of these cells. Our study revealed a physiologically significant role for PACT in cell proliferation and an essential role of a dsRNA-binding protein in mammalian pituitary expansion.

Biochemical Analysis of PKR Activation by PACT

Many extracellular stresses cause inhibition of translation initiation by triggering phosphorylation of the initiation factor, eIF-2alpha. A major protein kinase responsible for this phosphorylation is PKR, a latent kinase which itself needs to be activated by autophosphorylation. In stressed cells, this activation occurs when PACT, a PKR-binding protein, is phosphorylated and activates PKR. We have previously demonstrated that the presence of specific residues in domain 3 of PACT is necessary for its ability to activate PKR in vivo. Here, we analyze the biochemical properties of the inactive PACT mutants by assessing their ability to bind and activate PKR in vitro. Among the essential residues, two serines need to be phosphorylated in vivo for PACTs ability to activate PKR. We substituted those serines with aspartic acids, mimics of phosphoserines, and investigated the properties of the corresponding mutant PACTs. In vitro, they activate PKR more efficiently because they bind to PKR more tightly. These results indicate that stress-induced phosphorylation of specific serine residues in domain 3 of PACT increases its affinity for PKR, which leads to better activation of PKR and resultant eIF-2alpha phosphorylation.