Modification of the Stat1 SH2 Domain Broadly Improves Interferon Efficacy in Proportion to p300/CREB-binding Protein Coactivator Recruitment

Yong Zhang,Kazutaka Takami,Mindy S Lo,Guangming Huang,Qing Yu,William T Roswit,Michael J Holtzman

doi:10.1074/jbc.m503263200

Yong Zhang, Kazutaka Takami + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m503263200

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Abstract

A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference inspontaneousdimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-beta (that depends on Stat1/Stat2 heterodimerization) as well as IFN-gamma (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-gamma. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy.

Highlights

Against cancer [1, 2]
The present study demonstrates that specific modification of the Stat1 Src homology 2 (SH2) domain can markedly increase responsiveness to both type I and II IFN and defines the mechanism for this action
We show that mutagenesis of Stat1 SH2 domain has several unexpected functional consequences: (i) preservation of ligand-dependent activation, (ii) increased capacity for Tyr-701 phosphorylation and DNA binding, and (iii) enhanced recruitment of p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex

Summary

Introduction

Against cancer [1, 2]. The protective actions of IFN rely on signaling through two IFN receptors (IFNAR for type I and IFNGR for type II IFNs, respectively) and the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway that includes receptor-associated JAK and STAT as well as downstream transcription factors, enhancers, and co-activators [3]. Analogous to the findings in neoplasia, Stat activation in asthma developed despite normal tissue levels of IFN and so was labeled as constitutive In both conditions, the teleological basis for altered Stat behavior under pathologic conditions is uncertain but may represent an attempt to improve immune surveillance. We recognized that others have used crystal structures for Stat, Stat, and Stat as a guide (19 –21) to engineer a recombinant Stat protein with doublecysteine substitutions to enable spontaneous homodimerization (by disulfide linkage) that allows ligand- and phosphorylation-independent DNA binding and transcriptional activity in transduced cell lines [22] This mechanism has been assumed in follow-up studies of Stat as well as Stat function [23,24,25,26,27]. The apparent hyperresponsiveness of Stat found in inflammatory disease may represent an attempt to evolve Stat signaling capacity in this natural setting as well

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