Activation of Tyk2 and Stat3 is required for the apoptotic actions of interferon-beta in primary pro-B cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.     

Novel functions of tyrosine kinase 2 in the antiviral defense against murine cytomegalovirus

We have recently reported that tyrosine kinase 2 (Tyk2)-deficient mice have a selective defect in the in vivo defense against certain viruses. In our current study we show that Tyk2 is essential for the defense against murine CMV (MCMV). In vivo challenges with MCMV revealed impaired clearance of virus from organs and decreased survival of mice in the absence of Tyk2. Our in vitro studies demonstrate that MCMV replicates to dramatically higher titers in Tyk2-deficient macrophages compared with wild-type cells. We show an essential role of type I IFN (IFN-alphabeta) in the control of MCMV replication, with a prominent role of IFN-beta. MCMV infection leads to the activation of STAT1 and STAT2 in an IFN-alphabeta receptor 1-dependent manner. Consistent with the role of Tyk2 in IFN-alphabeta signaling, activation of STAT1 and STAT2 is reduced in Tyk2-deficient cells. However, lack of Tyk2 results in impaired MCMV-mediated gene induction of only a subset of MCMV-induced IFN-alphabeta-responsive genes. Taken together, our data demonstrate a requirement for Tyk2 in the in vitro and in vivo antiviral defense against MCMV infection. In addition to the established role of Tyk2 as an amplifier of Jak/Stat signaling upon IFN-alphabeta stimulation, we provide evidence for a novel role of Tyk2 as a modifier of host responses.     

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.     

Catalytically active TYK2 is essential for interferon-beta-mediated phosphorylation of STAT3 and interferon-alpha receptor-1 (IFNAR-1) but not for activation of phosphoinositol 3-kinase.

TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891-1897) that catalytically active TYK2 was necessary for IFN-beta to induce the beta-R1 gene. We now report IFN-beta-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-beta induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-beta-mediated phosphorylation of interferon-alpha receptor-1 (IFNAR-1) was defective in IFN-beta treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-beta treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-beta-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells.     

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.     

Tyrosine kinase 2 interacts with and phosphorylates receptor for activated C kinase-1, a WD motif-containing protein

Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.     

GTK tyrosine kinase-induced alteration of IRS-protein signalling in insulin producing cells

BACKGROUND: Insulin receptor substrate proteins (IRS) mediate various effects of insulin, including regulation of glucose homeostasis, cell growth and survival. To understand the underlying mechanisms explaining the effects of the Src-related tyrosine kinase GTK on beta-cell proliferation and survival, insulin-signalling pathways involving IRS-1 and IRS-2 were studied in islet cells and RINm5F cells overexpressing wild-type and two different mutants of the SRC-related tyrosine kinase GTK. MATERIALS AND METHODS: Islets isolated from transgenic mice and RINm5F cells overexpressing wild-type and mutant GTK were analysed for IRS-1, IRS-2, SHB, AKT and ERK phosphorylation/activity by Western blot analysis. RESULTS: RINm5F cells expressing the kinase active mutant Y504F-GTK and islet cells from GTK(Y504F) -transgenic mice exhibited reduced insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2. In RINm5F cells, the diminished IRS-phosphorylation was accompanied by a reduced insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K), AKT and Extracellular Signal-Regulated Kinase, partly due to an increased basal activity. In addition, increased tyrosine phosphorylation of the SHB SH2 domain-adaptor protein and its association with IRS-2, IRS-1 and focal adhesion kinase was observed in these cells. RINm5F cells overexpressing wild-type GTK also exhibited reduced activation of IRS-2, PI3K and AKT, whereas cells expressing a GTK mutant with lower kinase activity (GTK(Y394F)) exhibited insignificantly altered responses to insulin compared to the mock transfected cells. Moreover, GTK was shown to associate with and phosphorylate SHB in transiently transfected COS-7 cells, indicating that SHB is a specific substrate for GTK. CONCLUSIONS: The results suggest that GTK signals via SHB to modulate insulin-stimulated pathways in beta cells and this may explain previous results showing an increased beta-cell mass in GTK-transgenic mice.     

Protein-tyrosine phosphatase PTPepsilon C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells

We engineered and expressed both a wild-type and mutant cytosolic isoform of PTPepsilon (PTPepsilonC) in murine M1 leukemic cells, which can be induced to growth arrest and monocytic differentiation by interleukin (IL)-6 and leukemia inhibitory factor (LIF). Forced expression of PTPepsilonC inhibited IL-6- and LIF-induced monocytic differentiation and apoptosis in M1 cells, whereas expression of PTPepsilonM, a transmembrane isoform of PTPepsilon, did not. PTPepsilonC expression resulted in lower levels of IL-6-induced tyrosine phosphorylation of Jak1, Tyk2, gp130, and Stat3 compared with parent cells. In M1 transfectants expressing an inactive mutant of PTPepsilonC, both tyrosine phosphorylation and apoptosis induced by IL-6 and LIF were potentiated rather than inhibited. These results suggest an important role for PTPepsilonC in negative regulation of IL-6- and LIF-induced Jak-STAT signaling.     

Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells

Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn(-/-) mice (Lyn(-/-) BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn(-/-) BMMCs release more beta-hexosaminidase than wild-type BMMCs following FcepsilonRI cross-linking and show that multiple mast cell responses to FcepsilonRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cgamma isoforms, the mobilization of Ca(2+), the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the alpha(4)beta(1) integrin, VLA-4) are slow to initiate in Lyn(-/-) BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn(-/-) BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn(-/-) BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn(-/-) BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcepsilonRI signaling in Lyn(-/-) BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn(-/-) BMMCs may additionally contribute to their altered signaling properties.