Jennifer A. Young

PTPH1 Is a Predominant Protein-tyrosine Phosphatase Capable of Interacting with and Dephosphorylating the T Cell Receptor ζ Subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor ζ subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor ζ subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR ζ substrate (specific activity ranging from 0.23 to 40 pmol/min/μg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-ζ. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ζ ITAMs, with PTPH1 directly dephosphorylating ζ. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR ζ.

The formation and functions of the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits.

The interaction between the T cell receptor (TCR) and its cognate antigen/major histocompatibility complex (MHC) complex activates a cascade of intracellular protein phosphorylations within the T cell. The signals are initiated by the specific phosphorylation of two tyrosine residues located in a conserved sequence motif termed an ITAM (immune receptor‐based tyrosine activation motif). There are 10 ITAMs in the TCR complex, and 6 of these ITAMs are present in the TCR ζ homodimer. Following TCR stimulation, the TCR ζ subunit forms two tyrosine‐phosphorylated intermediates of 21‐ and 23‐kDa, respectively. The dramatic and diverse biological responses of T cells are proposed to be partly regulated by the relative ratios of the 21‐ vs. 23‐kDa phosphorylated forms of TCR ζ that are induced following TCR ligation. In this review, we describe a stepwise model of ζ phosphorylation required for the formation of these two phosphorylated derivatives. We describe the kinases and phosphatases controlling these phosphorylation processes. In addition, we present some preliminary findings from ongoing studies that discuss the contributions of each phosphorylated form of ζ on T cell development, TCR signaling, T cell anergy induction, and T cell survival.

Fas-associated Death Domain (FADD) and the E3 Ubiquitin-Protein Ligase TRIM21 Interact to Negatively Regulate Virus-induced Interferon Production

The production of cytokines such as type I interferon (IFN) is an essential component of innate immunity. Insufficient amounts of cytokines lead to host sensitivity to infection, whereas abundant cytokine production can lead to inflammation. A tight regulation of cytokine production is, thus, essential for homeostasis of the immune system. IFN-α production during RNA virus infection is mediated by the master transcription factor IRF7, which is activated upon ubiquitination by TRAF6 and phosphorylation by IKKϵ and TBK1 kinases. We found that Fas-associated death domain (FADD), first described as an apoptotic protein, is involved in regulating IFN-α production through a novel interaction with TRIM21. TRIM21 is a member of a large family of proteins that can impart ubiquitin modification onto its cellular targets. The interaction between FADD and TRIM21 enhances TRIM21 ubiquitin ligase activity, and together they cooperatively repress IFN-α activation in Sendai virus-infected cells. FADD and TRIM21 can directly ubiquitinate IRF7, affect its phosphorylation status, and interfere with the ubiquitin ligase activity of TRAF6. Conversely, a reduction of FADD and TRIM21 levels leads to higher IFN-α induction, IRF7 phosphorylation, and lower titers of RNA virus of infected cells. We conclude that FADD and TRIM21 together negatively regulate the late IFN-α pathway in response to viral infection.

The membrane-proximal portion of CD3 associates with the serine/threonine kinase GRK2

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 ϵ. Mass spectrometry analysis of CD3 ϵ-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 ϵ within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the α/β TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 ϵ may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.

The CD3 γε/δε signaling module provides normal T cell functions in the absence of the TCR ζ immunoreceptor tyrosine-based activation motifs

T cell receptor (TCR) signal transduction is mediated by the immunoreceptor tyrosine‐based activation motifs (ITAM). The ten ITAM in the TCR complex are distributed in two distinct signaling modules termed TCR ζζ and CD3 γϵ/δϵ. To delineate the specific role of the ζ ITAM in T cell development and TCR signal transmission, we compared the properties of T cells from different TCR ζ‐transgenic lines wherein tyrosine‐to‐phenylalanine substitutions had been introduced in the ζ subunit. These lines lack selected phosphorylated forms of TCR ζ including just p23, both p21 and p23, or all phospho‐ζ derivatives. We report herein that the efficiency of positive selection in HY TCR‐transgenic female mice was directly related to the number of ζ ITAM in the TCR. In contrast, TCR‐mediated signal transmission and T cell proliferative responses following agonist peptide stimulation were similar and independent of the ζ ITAM. Only the duration of MAPK activation was affected by multiple ζ ITAM substitutions. These results strongly suggest that the ITAM in the CD3 γϵ/δϵ module can provide normal TCR signal transmission, with ζ ITAM providing a secondary function facilitating MAPK activation and positive selection.

Commensal Microbiota Are Required for Systemic Inflammation Triggered by Necrotic Dendritic Cells

The relationship between dendritic cells (DCs) and commensal microflora in shaping systemic immune responses is not well understood. Here, we report that mice deficient for the Fas-associated death domain in DCs developed systemic inflammation associated with elevated proinflammatory cytokines and increased myeloid and B cells. These mice exhibited reduced DCs in gut-associated lymphoid tissues due to RIP3-dependent necroptosis, whereas DC functions remained intact. Induction of systemic inflammation required DC necroptosis and commensal microbiota signals that activated MyD88-dependent pathways in other cell types. Systemic inflammation was abrogated with the administration of broad-spectrum antibiotics or complete, but not DC-specific, deletion of MyD88. Thus, we have identified a previously unappreciated role for commensal microbiota in priming immune cells for inflammatory responses against necrotic cells. These studies demonstrate the impact intestinal microflora have on the immune system and their role in eliciting proper immune responses to harmful stimuli.

The protein tyrosine phosphatase PTPN4/PTP-MEG1. an enzyme capable of dephosphorylating the TCR ITAMs and regulating NF-κB, is dispensable for T cell development and/or T cell effector functions

T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR zeta subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-kappaB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogenes were equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations.

Haemophilus ducreyi Targets Src Family Protein Tyrosine Kinases To Inhibit Phagocytic Signaling

ABSTRACT Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcγ receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacte-rial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

Selective Expression of the 21-Kilodalton Tyrosine-Phosphorylated Form of TCR ζ Promotes the Emergence of T Cells with Autoreactive Potential

T cells undergo negative selection in the thymus to eliminate potentially autoreactive cells. The signals generated through the αβ TCR following receptor interactions with peptide/MHC complexes in the thymus control these selection processes. Following receptor ligation, a fraction of the TCR ζ subunit appears as two distinct tyrosine-phosphorylated forms of 21 and 23 kDa (p21 and p23). Previous data have reported elevated levels of p21 in some murine models of autoimmunity. We have examined the contributions of both the p21 and p23 to T cell negative selection in the HY TCR-transgenic system using ITAM-substituted TCR ζ and CD3 ε transgenic mice. Expression of just p21, in the absence of p23, partially impairs negative selection of self-reactive HY-specific T cells. This results in the emergence of potentially autoreactive peripheral T cells and an elevated population of CD11b+B220+ B cells in the spleen. These data clearly identify a specific and unique role for p21 during negative selection.

Deletion of FADD in macrophages and granulocytes results in RIP3- and MyD88-dependent systemic inflammation.

Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.