Ninan Abraham

Characterization of Transgenic Mice with Targeted Disruption of the Catalytic Domain of the Double-stranded RNA-dependent Protein Kinase, PKR

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor α-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targetedPkr-null cells. The observation of intact eukaryotic initiation factor-2α phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2α kinase(s).

The Murine Double-Stranded RNA-Dependent Protein Kinase PKR Is Required for Resistance to Vesicular Stomatitis Virus

ABSTRACT Interferon (IFN)-induced antiviral responses are mediated through a variety of proteins, including the double-stranded RNA-dependent protein kinase PKR. Here we show that fibroblasts derived from PKR−/− mice are more permissive for vesicular stomatitis virus (VSV) infection than are wild-type fibroblasts and demonstrate a deficiency in alpha/beta-IFN-mediated protection. We further show that mice lacking PKR are extremely susceptible to intranasal VSV infection, succumbing within days after instillation with as few as 50 infectious viral particles. Again, alpha/beta-IFN was unable to rescue PKR−/− mice from VSV infection. Surprisingly, intranasally infected PKR−/− mice died not from pathology of the central nervous system but rather from acute infection of the respiratory tract, demonstrating high virus titers in the lungs compared to similarly infected wild-type animals. These results confirm the role of PKR as the major component of IFN-mediated resistance to VSV infection. Since previous reports have shown PKR to be nonessential for survival in animals challenged with encephalomyocarditis virus, influenza virus, and vaccinia virus (N. Abraham et al., J. Biol. Chem. 274:5953–5962, 1999; Y. Yang et al., EMBO J. 14:6095–6106, 1995), our findings serve to highlight the premise that host dependence on the various mediators of IFN-induced antiviral defenses is pathogen specific.

Double-Stranded-RNA-Activated Protein Kinase PKR Enhances Transcriptional Activation by Tumor Suppressor p53

ABSTRACT The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR+/+ and PKR−/− mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G0/G1 arrest were impaired in PKR−/− cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser18 was defective in PKR−/− cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21WAF/Cip1 and Mdm2. In addition, Ser18 phosphorylation and transcriptional activation by mouse p53 were diminished in PKR−/− cells after DNA damage induced by the anticancer drug adriamycin or γ radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser18 of p53 by adriamycin to a higher degree in PKR+/+ cells than in PKR−/− cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.

Loss of Tolerance and Autoimmunity Affecting Multiple Organs in STAT5A/5B-Deficient Mice

STAT5 has previously been reported to be dispensable for the maintenance of tolerance in vivo. However, in examining hemopoiesis in mice lacking both isoforms of STAT5, STAT5A, and STAT5B, we noted that a subset of these mice demonstrated dramatic alterations in several bone marrow progenitor populations concomitant with lymphocytic infiltration of the bone marrow. In addition, cellular infiltration affecting the colon, liver, and kidney was observed in these mice. Survival analysis revealed that STAT5A/5B−/− mice exhibited early death. The increased mortality and the pathology affecting multiple organs observed in these mice were abrogated on the recombination-activating gene 1−/− background. In light of the similarities between STAT5A/5B-deficient mice and mice unable to signal through the IL-2R, we hypothesized that the tolerizing role of STAT5A/5B was triggered via activation of the IL-2R. In agreement with this, we found that IL-2Rβ chain-deficient mice exhibited similar hemopoietic abnormalities. Because IL-2 signaling is thought to contribute to tolerance through maintenance of a CD4+CD25+ regulatory T cell population, we examined these cells and observed a numerical reduction in STAT5A/5B−/− mice along with a higher rate of apoptosis. These data provide strong evidence for a requirement for STAT5 in the maintenance of tolerance in vivo.

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.

Impaired CD8 T cell memory and CD4 T cell primary responses in IL-7Rα mutant mice

Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Rα, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Rα Y449XXM motif in mice by knock-in mutagenesis (IL-7Rα449F). Thymic precursors were reduced in number in IL-7Rα449F mice, but in marked contrast to IL-7Rα−/− knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Rα signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Rα449F mice. Furthermore, we show that Bcl-2 is IL-7Rα Y449 independent and insufficient for IL-7–mediated maintenance of CD8 memory.

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

To understand the mechanism(s) by which p56lck participates in T-cell receptor (TCR) signalling, we have examined the effects of mutations in known regulatory domains of p56lck on the ability of F505 p56lck to enhance the responsiveness of an antigen-specific murine T-cell hybridoma. A mutation of the amino-terminal site of myristylation (glycine 2), which prevents stable association of p56lck with the plasma membrane, completely abolished the ability of F505 p56lck to enhance TCR-induced tyrosine protein phosphorylation. Alteration of the major site of in vitro autophosphorylation, tyrosine 394, to phenylalanine diminished the enhancement of TCR-induced tyrosine protein phosphorylation by F505 p56lck. Such a finding is consistent with the previous demonstration that this site is required for full activation of p56lck by mutation of tyrosine 505. Strikingly, deletion of the noncatalytic Src homology domain 2, but not of the Src homology domain 3, markedly reduced the improvement of TCR-induced tyrosine protein phosphorylation by F505 Lck. Additional studies revealed that all the mutations tested, including deletion of the Src homology 3 region, abrogated the enhancement of antigen-triggered interleukin-2 production by F505 p56lck, thus implying more stringent requirements for augmentation of antigen responsiveness by F505 Lck. Finally, it was also observed that expression of F505 p56lck greatly increased TCR-induced tyrosine phosphorylation of phospholipase C-gamma 1, raising the possibility that phospholipase C-gamma 1 may be a substrate for p56lck in T lymphocytes. Our results indicate that p56lck regulates T-cell antigen receptor signalling through a complex process requiring multiple distinct structural domains of the protein.

Mucosal memory CD8 + T cells are selected in the periphery by an MHC class I molecule

The presence of immune memory at pathogen-entry sites is a prerequisite for protection. Nevertheless, the mechanisms that warrant immunity at peripheral interfaces are not understood. Here we show that the nonclassical major histocompatibility complex (MHC) class I molecule thymus leukemia antigen (TL), induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells, mediated affinity-based selection of memory precursor cells. Furthermore, constitutive expression of TL on epithelial cells led to continued selection of mature CD8αβ+ memory T cells. The memory process driven by TL and CD8αα was essential for the generation of CD8αβ+ memory T cells in the intestine and the accumulation of highly antigen-sensitive CD8αβ+ memory T cells that form the first line of defense at the largest entry port for pathogens.

Regulation of memory T cells by γc cytokines.

T cells rely on a duality of TCR and gammac cytokine signals for development, activation and peripheral T cell homeostasis. Previous data had suggested that the requirements for CD4 and CD8 memory T cell regulation were qualitatively distinct, but emerging data has shown that the requirements for true antigen specific memory T cells are very similar between these two cell types. This review will focus on contributions made by members of the gammac cytokine family (IL-2, IL-4, IL-7, IL-15 and IL-21) to homeostasis of naïve, memory phenotype and antigen experienced memory T cells.

Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas.

The requirement for receptor components and the signalling effector, signal transducer and activator of transcription (STAT) 5A/5B, was assessed genetically in a lymphoma development model induced by interleukin-7 (IL-7). This growth factor for T- and B-cell progenitors and mature lymphocytes activates survival and proliferative pathways including Bcl-2, phosphatidylinositol-3 kinase and STAT5. Overexpression of IL-7 in vivo causes early mortality from lymphoma development. Mice overexpressing IL-7 that were heterozygous for the IL-7Rα subunit showed improved survival compared to wild-type mice. In addition, STAT5A/5B+/− compound heterozygous mice with one targeted allele each of STAT5A and STAT5B showed striking amelioration of IL-7-induced mortality and disease development. STAT5A/5B+/− compound heterozygous mice were otherwise normal in stem cell and lymphocyte development and cellularity. Lower STAT5 protein levels accompanied the reduction in STAT5A/5B copy number, which suggests that STAT5 haploinsufficiency is a modifier of IL-7 signal strength.