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A Phosphosite Screen Identifies Autocrine TGF-β-Driven Activation of Protein Kinase R as a Survival-Limiting Factor for Eosinophils¹

Nicholas Goplen^*, Magdalena M. Gorska^*,, Susan J. Stafford, Sadee Rozario^*, Lei Guo^*, Qiaoling Liang^* and Rafeul Alam^2,*,

^* National Jewish Medical and Research Center, Denver, CO 80206; University of Colorado at Denver Health Sciences Center, Denver, CO 80206; and University of Texas Medical Branch, Galveston, TX 77555

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase C, and glycogen synthase kinase 3 and β. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2 are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-β in eosinophils. TGF-β directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-β Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-β and that this could be modulated by a Th2 microenvironment in vivo.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants R01 AI50179, AI059719, and AI68088.

² Address correspondence and reprint requests to Dr. Rafeul Alam, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: alamr{at}njrc.org

³ Abbreviations used in this paper: EIF2, eukaryotic initiation factor 2; DAPI, 4',6'-diamidino-2-phenylindole; PVDF, polyvinylidene difluoride; IFS, immunofluorescent staining; GSK3, glycogen synthase kinase 3; PKR, protein kinase R; NT, nontargeting; siRNA, small interfering RNA; MSK, mitogen and stress activated kinase; RSK, ribosomal S6 kinase.