Darren P. Baker

Identification of a Palmitic Acid-modified Form of Human Sonic hedgehog

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24–197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the α-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

Function of Mitochondrial Stat3 in Cellular Respiration

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3–/– cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the proteins function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors.

Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial/mesenchymal interactions during embryonic development. We found that the hedgehog-signaling pathway is present in adult cardiovascular tissues and can be activated in vivo. Shh was able to induce robust angiogenesis, characterized by distinct large-diameter vessels. Shh also augmented blood-flow recovery and limb salvage following operatively induced hind-limb ischemia in aged mice. In vitro, Shh had no effect on endothelial-cell migration or proliferation; instead, it induced expression of two families of angiogenic cytokines, including all three vascular endothelial growth factor-1 isoforms and angiopoietins-1 and -2 from interstitial mesenchymal cells. These findings reveal a novel role for Shh as an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders.

Comparative biological responses to human Sonic, Indian, and Desert hedgehog.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

Role of the Akt pathway in mRNA translation of interferon-stimulated genes

Multiple signaling pathways are engaged by the type I and II IFN receptors, but their specific roles and possible coordination in the generation of IFN-mediated biological responses remain unknown. We provide evidence that activation of Akt kinases is required for IFN-inducible engagement of the mTOR/p70 S6 kinase pathway. Our data establish that Akt activity is essential for up-regulation of key IFN-α- and IFN-γ-inducible proteins, which have important functional consequences in the induction of IFN responses. Such effects of the Akt pathway are unrelated to regulatory activities on IFN-dependent STAT phosphorylation/activation or transcriptional regulation. By contrast, they reflect regulatory activities on mRNA translation via direct control of the mTOR pathway. In studies using Akt1 and Akt2 double knockout cells, we found that the absence of Akt kinases results in dramatic reduction in IFN-induced antiviral responses, establishing a critical role of the Akt pathway in IFN signaling. Thus, activation of the Akt pathway by the IFN receptors complements the function of IFN-activated JAK–STAT pathways, by allowing mRNA translation of IFN-stimulated genes and, ultimately, the induction of the biological effects of IFNs.

IFN-α and IFN-λ differ in their antiproliferative effects and duration of JAK/STAT signaling activity

Interferon (IFN)-λ, also known as IL-28A, IL-28B or IL-29, is a new type III IFN, which like type I IFN-(α/β), activates common elements of the JAK/STAT signaling pathway and exhibits antiproliferative activity. Currently, IFN-α is used in the treatment of certain forms of cancer, but its antitumor effects are limited and associated with high toxicity. In this study, we determined whether IFN-λ induced the same level of cell growth inhibition relative to IFN-α. To this effect HaCaT cells, which are typically growth inhibited by IFN-α, underwent apoptosis in response to IFN-λ. Next, in contrast to IFN-α stimulation, IFN-λ prolonged the duration of activated STAT1 and STAT2. Furthermore, the kinetics of IFN-stimulated genes was different as IFN-λ induced a delayed but stronger induction of IFN-responsive genes. Components of the JAK/STAT pathway remained essential for the antiproliferative effects of IFN-α and IFN-λ. IFN-λ-induced persistence of STAT activation required de novo protein synthesis and was in part due to a delay in STAT2 inactivation. Thus our data demonstrate that the duration of IFN-λ signaling is different from that of IFN-α, and that IFN-λ could be a suitable cytokine to evaluate for cancer therapy.

Cutting Edge: Role of STAT1, STAT3, and STAT5 in IFN-αβ Responses in T Lymphocytes

Engagement of the IFN-αβ receptor initiates multiple signaling cascades, including activation of the STAT. In this study, we demonstrate that IFN-αβ, although antiproliferative in wild-type CD4+ or CD8+ T cells, act as strong mitogens on their STAT1−/− counterparts. Furthermore, IFN-αβ exert little effect on apoptosis in wild-type cells, but are potent survival factors in the absence of STAT1. The antiapoptotic response in the absence of STAT1 is predominantly mediated by STAT3, and to a lesser extent by STAT5A/B. In contrast, the mitogenic IFN-αβ response gained through the absence of STAT1 is only marginally affected when STAT5A/B expression is also abrogated, but is completely dependent on STAT3 activation. These findings provide the first evidence for a function of STAT3 and STAT5A/B in the IFN-αβ response, and support a model in which the IFN-αβ receptor initiates both pro- and antiapoptotic responses through STAT1, and STAT3 and STAT5A/B, respectively.

Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Regulatory Effects of Mammalian Target of Rapamycin-activated Pathways in Type I and II Interferon Signaling

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNα or IFNγ results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNα- or IFNγ-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1-/-) knockout MEFs, as compared with wild-type 4E-BP1+/+ MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2-/-) or hamartin (TSC1-/-) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.

Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.