Proteasome-mediated Degradation of STAT1α following Infection of Macrophages with Leishmania donovani

David J Gregory,Geneviève Forget,Martin Olivier

doi:10.1074/jbc.m414126200

Abstract

Activation of the Janus-activated kinase 2 (JAK2)/STAT1alpha signaling pathway is repressed in Leishmania-infected macrophages. This represents an important mechanism by which this parasite subverts the microbicidal functions of the cell to promote its own survival and propagation. We recently provided evidence that the protein tyrosine phosphatase (PTP) SHP-1 was responsible for JAK2 inactivation. However, STAT1 translocation to the nucleus was not restored in the absence of SHP-1. In the present study, we have used B10R macrophages to study the mechanism by which this Leishmania-induced STAT1 inactivation occurs. STAT1alpha nuclear localization was shown to be rapidly reduced by the infection. Western blot analysis revealed that cellular STAT1alpha, but not STAT3, was degraded. Using PTP inhibitors and an immortalized bone marrow-derived macrophage cell line from SHP-1-deficient mice, we showed that STAT1 inactivation was independent of PTP activity. However, inhibition of macrophage proteasome activity significantly rescued Leishmania-induced STAT1alpha degradation. We further demonstrated that degradation was receptor-mediated and involved protein kinase C alpha. All Leishmania species tested (L. major, L. donovani, L. mexicana, L. braziliensis), but not the related parasite Trypanosoma cruzi, caused STAT1alpha degradation. Collectively, results from this study revealed a new mechanism for STAT1 regulation by a microbial pathogen, which favors its establishment and propagation within the host.

Highlights

Intracellular protozoan parasites of the Leishmania genus are the etiological agents of leishmaniasis, a condition that causes considerable worldwide morbidity and mortality, with pathologies ranging from disfiguring cutaneous to lethal visceral afflictions [1]
We have previously shown that the unresponsiveness of the Janus-activated kinase 2 (JAK2) signaling pathway in Leishmania donovani-infected macrophages upon IFN␥ stimulation was due to the ability of the parasite to activate a particular protein tyrosine phosphatase (PTP), SHP-1 [4]
Effect of L. donovani Infection on STAT1␣ Nuclear Localization—Previous studies have reported that JAK2/STAT1 signaling is altered in Leishmania-infected macrophages [4, 6]

Summary

The abbreviations used are

IFN␥, interferon ␥; CR3, complement receptor 3; EMSA, electrophoretic mobility shift assay; GAS/ISRE, ␥-activated sequence/interferon stimulation response element; JAK, Janusactivated kinase; PKC, protein kinase C; PTP, protein tyrosine phosphatase; PIAS-1, protein inhibitor of activated STAT1; SOCS-1, suppressor of cytokine signalling-1; STAT, signal transducer and activator of transcription; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bpV(phen), potassium bispexoxol(1, 10phenanthroline) oxovanadate. Other groups have reported that this degradation is celltype-specific and suggested that inhibition of STAT1 phosphorylation is a more important mechanism [32, 36]. We investigated the mechanisms whereby Leishmania infection causes macrophage STAT1␣ inactivation. Our study further implicates protein kinase C␣ (PKC␣)-dependent signaling and proteasomes (but not PTPs) in this process. Together, these results argue for a direct role of the proteasome pathway in the specific proteolysis of STAT1␣ in macrophages infected by the protozoan parasite Leishmania, representing a new mechanism whereby microbial pathogens can subvert microbicidal activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION