Functional Immunoreceptor Tyrosine-based Activation Motif Research Articles

An ITAM in a nonenveloped virus regulates activation of NF-κB, induction of beta interferon, and viral spread.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: μ2, σ2, and λ2. We demonstrate for the first time that μ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, μ2 and μNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the μ2 ITAM. Moreover, both the μ2 ITAM and Syk are required for maximal μ2 activation of NF-κB. A mutant virus lacking the μ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-β) than does wild-type virus despite similar replication. Notably, the consequences of these μ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the μ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the μ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-β. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.

Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

SummaryThe physiological basis and mechanistic requirement for the high immunoreceptor tyrosine activation motifs (ITAM) multiplicity of the T cell receptor (TCR)-CD3 complex remains obscure. Here we show that while low TCR-CD3 ITAM multiplicity is sufficient to engage canonical TCR-induced signaling events that lead to cytokine secretion, high TCR-CD3 ITAM multiplicity is required for TCR-driven proliferation. This is dependent on compact immunological synapse formation, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate Notch1 recruitment and activation and ultimately c-Myc-induced proliferation. Analogous mechanistic events are also required to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and co-ordinated by the multiplicity of phosphorylated TCR-CD3 ITAMs.

An Immunoreceptor Tyrosine Activation Motif in the Mouse Mammary Tumor Virus Envelope Protein Plays a Role in Virus-Induced Mammary Tumors

Mouse mammary tumor virus (MMTV) induces breast cancer with almost 100% efficiency in susceptible strains through insertional activation of protooncogenes, such as members of the wnt and fibroblast growth factor (fgf) families. We previously showed that expression of the MMTV envelope protein (Env) in normal immortalized mammary epithelial cells grown in three-dimensional cultures caused their morphological transformation, and that this phenotype depended on an immunoreceptor tyrosine-based activation motif (ITAM) present in Env and signaling through the Syk tyrosine kinase (E. Katz, M. H. Lareef, J. C. Rassa, S. M. Grande, L. B. King, J. Russo, S. R. Ross, and J. G. Monroe, J. Exp. Med. 201:431-439, 2005). Here, we examined the role of the Env protein in virus-induced mammary tumorigenesis in vivo. Similar to the effect seen in vitro, Env expression in the mammary glands of transgenic mice bearing either full-length wild-type provirus or only Env transgenes showed increased lobuloalveolar budding. Introduction of the ITAM mutation into the env of an infectious, replication-competent MMTV or into MMTV/murine leukemia virus pseudotypes had no effect on incorporation of Env into virus particles or on in vitro infectivity. Moreover, replication-competent MMTV bearing the ITAM mutation in Env infected lymphoid and mammary tissue at the same level as wild-type MMTV and was transmitted through milk. However, mammary tumor induction was greatly attenuated, and the pattern of oncogene activation was altered. Taken together, these studies indicate that the MMTV Env protein participates in mammary epithelial cell transformation in vivo and that this requires a functional ITAM in the envelope protein.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Modulates K1 Expression through Its cis -Acting Elements within the Terminal Repeats

K1 is the first open reading frame encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and lies positionally to the immediate right of the terminal repeats. K1 is a transmembrane glycoprotein having a functional immunoreceptor tyrosine-based activation motif (ITAM) capable of activating B-cell receptor signaling. K1 is expressed mostly during the lytic cycle of the virus and its promoter lies within the terminal repeat which contains the binding sites for latency-associated nuclear antigen (LANA). The K1 promoter (K1p) having LANA binding sites assayed by reporter assay demonstrated that LANA is capable of down-regulating K1 promoter transcriptional activity. However, the KSHV replication transcription activator RTA up-regulates K1p transcriptional activity. The promoter deleted of LANA binding sites showed loss in LANA-mediated down-regulation but was unaffected for RTA-mediated up-regulation. Increasing amounts of RTA rescued LANA-mediated repression of K1p transcriptional activity in cotransfection experiments. Reporter assay data suggest that LANA binding to its cognate sequence is critical for LANA-mediated repression of K1p as a LANA construct lacking the DNA binding domain was unable to repress K1p transcription. Additionally, KSHV primary infection experiments suggest that K1 is expressed during early infection but is repressed on the establishment of latency and so follows an expression profile similar to that of RTA during infection. Analysis of the promoter sequence revealed the presence of Oct-1 transcription factor binding sites within the -116 to +76 region. Mutational analysis of the Oct-1 sites abolished RTA-mediated transcriptional activation, suggesting that RTA up-regulates K1p transcription through binding to this transcription factor.

Cellular ITAM-containing proteins are oncoproteins in nonhematopoietic cells

Immunoreceptor tyrosine-based activation motifs (ITAMs) are involved in the transduction of signals necessary for activation, differentiation, and survival in hematopoietic cells. Several viruses have been shown to encode ITAM-containing transmembrane proteins. Although expression of these viral proteins has in some cases been shown to transform nonhematopoietic cells, a causal role for a functional ITAM in this process has not been elucidated. To examine the potential transforming properties of ITAM-containing proteins, a recombinant protein consisting of ITAM-containing cytoplasmic regions of the B-cell antigen receptor was expressed in immortalized murine mammary epithelial and fibroblast cells. Mammary epithelial cells expressing this construct exhibited depolarized morphology in three-dimensional cultures. This transformed phenotype was characterized by a loss of anchorage dependence and hallmarks of epithelial to mesenchymal transition. Fibroblasts expressing this ITAM construct also lost contact inhibition and anchorage dependence. The transformed phenotype seen in both cell types was abrogated upon tyrosine to phenylalanine substitutions of the ITAMs. Inhibition of Syk tyrosine kinase, which associates with the ITAM, also prevented cell transformation. Our results indicate that expression of a nonviral ITAM-containing protein is sufficient for cell transformation. Despite lacking intrinsic enzymatic activity, ITAM-containing proteins can function as potent oncoproteins by scaffolding downstream mediators.

Activation of Src kinase Lyn by the Kaposi sarcoma–associated herpesvirus K1 protein: implications for lymphomagenesis

AbstractThe K1 gene of Kaposi sarcoma–associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-κB) inhibitor Bay 11-7085 or an anti–vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-κB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence–deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-κB activation, indicating that ITAM sequences were required for the Lyn kinase–mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-κB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements.

Hantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.

Immunoreceptor tyrosine-based activation motif-dependent signaling by Kaposi's sarcoma-associated herpesvirus K1 protein: effects on lytic viral replication.

The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 gene encodes a polypeptide bearing an immunoreceptor tyrosine-based activation motif (ITAM) that is constitutively active for ITAM-based signal transduction. Although ectopic overexpression of K1 in cultured fibroblasts can lead to growth transformation, in vivo this gene is primarily expressed in lymphoid cells undergoing lytic infection. Here we have examined function of K1 in the setting of lytic replication, through the study of K1 mutants lacking functional ITAMs. Expression of such mutants in BJAB cells cotransfected with wild-type K1 results in dramatic inhibition of K1 signal transduction, as judged by impaired activation of Syk kinase and phospholipase C-gamma2 as well as by diminished expression of a luciferase reporter gene dependent upon K1-induced calcium and Ras signaling. Thus, the mutants behave as dominantly acting inhibitors of K1 function. To assess the role of K1 in lytic replication, we introduced these K1 mutants into BCBL-1 cells, a B-cell lymphoma line latently infected with KSHV, and induced lytic replication by ectopic expression of the KSHV ORF50 transactivator. Expression of lytic cycle genes was diminished up to 80% in the presence of a K1 dominant negative mutant. These inhibitory effects could be overridden by tetradecanoyl phorbol acetate treatment, indicating that inhibition was not due to irreversible cell injury and suggesting that other signaling events could bypass the block. We conclude that ITAM-dependent signaling by K1 is not absolutely required for lytic reactivation but functions to modestly augment lytic replication in B cells, the natural reservoir of KSHV.

Deregulated signal transduction by the K1 gene product of Kaposi's sarcoma-associated herpesvirus.

The Kaposi's sarcoma (KS)-associated herpesvirus is a lymphotropic virus strongly implicated in the pathogenesis of KS and several lymphoproliferative disorders. The KS-associated herpesvirus K1 gene encodes a transmembrane protein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM)-like sequence; it previously has been proposed to be important in viral tumorigenesis because its expression can trigger cell proliferation in vitro and in vivo. Here we show that expression of the full-length K1 protein can initiate calcium-dependent signal transduction in B cells; however, unlike other ITAM-based signal transduction events, K1 signaling occurs constitutively, in the absence of exogenous crosslinking ligands. This property is caused by its cysteine-rich ectodomain, which when transferred to other consensus ITAMs induces constitutive signaling. Although ITAM-based signaling by K1 involves classical syk and phospholipase C gamma2 activation, both ITAM- and syk-independent signaling pathways are activated by K1 expression. These studies indicate that K1 is a deregulated signaling molecule with pleitropic effects that may explain its known growth deregulatory properties.

Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

Activation of the T-cell receptor signaling pathway by Nef from an aggressive strain of simian immunodeficiency virus.

The Nef from a highly virulent strain of simian immunodeficiency virus (SIV), SIVpbj14, and a Nef from the traditional strain SIVmac239 bearing the mutation from RQ to YE (YE-Nef) both induce an acute lethal disease in monkeys. The YE mutation and its surrounding sequence resemble the immunoreceptor tyrosine-based activation motif (ITAM), which is present in the cytoplasmic tail of T- and B-cell antigen receptors and mediates signaling during lymphocyte activation. We show here that the ITAM from YE-Nef performs the same function. First, not only does YE-Nef increase the activity of the transcription factor NFAT, which is one of the downstream targets of T-cell activation, but the ITAM from the YE-Nef by itself also activates NFAT. Second, the ITAM from YE-Nef is phosphorylated on tyrosine residues by Lck and associates with ZAP-70, a T-cell-specific tyrosine kinase. The phosphorylation of both conserved tyrosine residues on the ITAM is required for the recruitment of ZAP-70. Finally, Lck is required for the activation of NFAT by YE-Nef. These results demonstrate that YE-Nef contains a functional ITAM and elucidate the molecular mechanisms underlying the pathogenesis of SIVpbj14.