Interaction of IL-2R beta and gamma c chains with Jak1 and Jak3: implications for XSCID and XCID.

Interleukin-2 (IL-2) signaling requires the dimerization of the IL-2 receptor beta.(IL-2R beta) and common gamma (gamma c) chains. Mutations of gamma c can result in X-linked severe combined immunodeficiency (XSCID). IL-2, IL-4, IL-7 (whose receptors are known to contain gamma c), and IL-9 (whose receptor is shown here to contain gamma c) induced the tyrosine phosphorylation and activation of the Janus family tyrosine kinases Jak1 and Jak3. Jak1 and Jak3 associated with IL-2R beta and gamma c, respectively; IL-2 induced Jak3-IL-2R beta and increased Jak3-gamma c associations. Truncations of gamma c, and a gamma c, point mutation causing moderate X-linked combined immunodeficiency (XCID), decreased gamma c-Jak3 association. Thus, gamma c mutations in at least some XSCID and XCID patients prevent normal Jak3 activation, suggesting that mutations of Jak3 may result in an XSCID-like phenotype.

Functional activation of Jak1 and Jak3 by selective association with IL-2 receptor subunits.

The interleukin-2 receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta, and IL-2R gamma chains, the last of which is also used in the receptors for IL-4, IL-7, and IL-9. Stimulation with IL-2 induces the tyrosine phosphorylation and activation of the Janus kinases Jak1 and Jak3. Jak1 and Jak3 were found to be selectively associated with the "serine-rich" region of IL-2R beta and the carboxyl-terminal region of IL-2R gamma, respectively. Both regions were necessary for IL-2 signaling. Furthermore, Jak3-negative fibroblasts expressing reconstituted IL-2R became responsive to IL-2 after the additional expression of Jak3 complementary DNA. Thus, activation of Jak1 and Jak3 may be a key event in IL-2 signaling.

Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components.

A recently defined family of cytokines, consisting of ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6), utilize the Jak-Tyk family of cytoplasmic tyrosine kinases. The beta receptor components for this cytokine family, gp130 and LIF receptor beta, constitutively associate with Jak-Tyk kinases. Activation of these kinases occurs as a result of ligand-induced dimerization of the receptor beta components. Unlike other cytokine receptors studied to date, the receptors for the CNTF cytokine family utilize all known members of the Jak-Tyk family, but induce distinct patterns of Jak-Tyk phosphorylation in different cell lines.

Defective lymphoid development in mice lacking Jak3.

The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common gamma (gamma c) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in gamma c signaling and lymphoid development.

Signaling by the cytokine receptor superfamily: JAKs and STATs.

A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.

Dentin Priming with Amphipathic Antimicrobial Peptides.

The evolution of bonded restorations has undergone great progress over several decades. Nonetheless, life spans of bonded restorations are limited mainly because of the eventual incidence of recurrent caries. Over time, water and waterborne agents (acids, enzymes) degrade the components of the dentin/restoration interface, allowing bacterial colonization and dentin reinfection at the margins of the restoration. We developed a 2-tier protective technology consisting of priming/coating dentin with amphipathic and antimicrobial peptides (AAMPs) to obtain hydrophobic/water-repellent and antibiofilm dentin-resisting recurrent caries around bonded restorations. We tested a series of AAMPs to assess their structure-function relationships as well as the effects of different dentin-conditioning methods on the structural features of AAMP-coated dentin. We found relation between the secondary structure of AAMPs (high portion of ß-sheet), the antimicrobial potency of AAMPs, and the AAMPs' ability to form hydrophobic coatings on dentin. We also determined that AAMPs had preferential adsorption on the mineral phase of dentin, which suggested that peptides arrange their cationic and hydrophilic motifs in direct contact with the negatively charged minerals in the hydrophilic dentin. These results led us to explore different dentin-conditioning methods that would increase the mineral/collagen ratio and their effect on AAMP immobilization. We innovatively imaged the spatial distribution of the AAMPs in relation to the dentinal tubules and collagen network using a minimally invasive multimodal imaging technique: multiphoton-second harmonic generation. Using multiphoton-second harmonic generation imaging, we determined that partial deproteinization of dentin increased the amount of immobilized AAMPs as compared with the total etched dentin at the dentin surface and extended deeply around dentinal tubules. Last, we analyzed the release rate of AAMPs from dentin coatings in artificial saliva to predict their stability in the clinical setting. In conclusion, priming dentin with AAMPs is a versatile new approach with potential to fortify the otherwise vulnerable adhesive-based interfaces.

Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Isolation and characterization of the rat beta-subunit gene of the high affinity receptor for immunoglobulin E.

The high affinity receptor for IgE, which mediates allergic reactions, is found exclusively on mast cells, basophils and epidermal Langerhans cells. It is composed of an alpha, beta and two gamma-polypeptide chains, inserted as a complex into the plasma membrane. The alpha and beta-subunits are produced only by these cell types. This work describes the isolation and characterization of the gene which encodes the rat beta-subunit. The single copy gene spans a 9 kbp region of DNA and is composed of seven exons and six introns. Transcriptional initiation begins from a single site, which is preceded by a very unique, putative transcriptional consensus sequence, the GATA box. Analysis of the 5'-region, upstream of exon 1, also reveals a unique sequence bias and a collection of repeating elements, suggesting several other potential transcriptional cis-control elements. The origins of the two, previously reported, different beta-subunit transcripts result from the same start site of this gene, but use an alternative RNA processing mechanism involving exon 3.

Tissue-specific expression in mouse P815 mastocytoma cells of the cloned rat alpha-subunit gene of the high-affinity receptor for immunoglobulin E.

The high-affinity multisubunit receptor for IgE, Fc epsilon RI, is expressed in vivo in a tissue-specific manner, being found only on mast cells, basophils and epidermal Langerhans cells. The expression of the rat Fc epsilon RI alpha-subunit transcript has been examined here in the mouse mastocytoma cell line, P815, which does not express the endogenous mouse Fc epsilon RI alpha- or beta-subunit transcripts. These studies indicate that an exogenously introduced rat alpha-subunit gene can be faithfully expressed in P815 cells, while no transcripts are produced in the mouse or rat B-lymphoid cell lines, SP2/0 and IR162, respectively. Therefore, in contrast to both B-cell lines, factors necessary for the tissue-specific expression of the alpha-subunit mRNA are present in the P815 cell line, yielding a correctly processed mRNA transcript; nevertheless, this tissue-specific expression is not rigorously species specific. Although the mechanism responsible for these observations is unknown, these results do imply that a specific cis-trans element interaction occurs between this transcriptional unit and factors in the P815 cells that control fidelity of tissue-specific mRNA synthesis and processing from the Fc epsilon RI alpha-subunit gene. Consequently, this reconstructed rat Fc epsilon RI alpha-subunit gene contains minimally sufficient cis elements for tissue-specific expression of mRNA, and so, the P815 cell line should be useful to define these requisite DNA cis elements of the gene, as well as the trans factors from P815 necessary for this process.

Differential substrate recognition capabilities of Janus family protein tyrosine kinases within the interleukin 2 receptor (IL2R) system: Jak3 as a potential molecular target for treatment of leukemias with a hyperactive Jak-Stat signaling machinery.

Substrate recognition by Janus family protein tyrosine kinases was examined utilizing recombinant baculovirus produced components of the interleukin 2 receptor (IL2R) system i.e. Jak1, Transducers and Activators of Transcription (STAT). Wild type Jak3 was able to tyrosine phosphorylate a kinase-dead Jak1 (Jak1E908). In contrast wild type Jak1 was unable to tyrosine phosphorylate kinase dead Jak3 (Jak3E851). This unilateral transphosphorylation between Jak3 and Jak1 prompts the hypothesis that in the IL2R system the activation of Jak3 precedes Jak1 activation. Both the IL2Rbeta and IL2Rgammac subunits underwent tyrosine phosphorylation when co-expressed with wild-type Jak3. By comparison only IL2Rbeta was recognized and tyrosine phosphorylated by wild-type Jak1. These results are consistent with the notion that Jakl is pre-associated with IL2Rbeta and Jak3 is pre-associated with IL2Rgammac. STAT1, STAT3, and STAT5 underwent tyrosine phosphorylation when co-expressed with Jakl and therefore are substrates for the respective Jak kinases. In contrast, Jak3 co-expression resulted in tyrosine phosphorylation of STAT3 and STAT5 but not STAT1. Notably a polypeptide representing the kinase domain of Jak3 (Jak3-JH1) gained the ability to tyrosine phosphorylate STAT1, suggesting that the changes in substrate recognition may be influenced by domains outside the kinase domain. These findings provide evidence that Jak1 and Jak3 differentially recognize specific substrates, thereby having the ability to contribute specific signals, and the substrate specificity may be influenced by multiple domains of these tyrosine kinases.

Wolbachia from the planthopper Laodelphax striatellus establishes a robust, persistent, streptomycin-resistant infection in clonal mosquito cells.

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales: Anaplasmataceae), distorts reproduction of its arthropod hosts to facilitate invasion of naïve populations. This property makes Wolbachia an attractive "gene drive" agent with potential applications in the control of insect vector populations. Genetic manipulation of Wolbachia will require in vitro systems for its propagation, genetic modification, amplification, and introduction into target insects. Here we show that Wolbachia from the planthopper, Laodelphax striatellus, establishes a robust infection in clonal C7-10 Aedes albopictus mosquito cells. Infected cells, designated C/wStr, expressed radiolabeled proteins that were enriched in cells grown in the absence of antibiotics that inhibit Wolbachia, relative to cultures grown in medium containing tetracycline and rifampicin. Using mass spectrometry, we verified that tryptic peptides from an upregulated 24 kDa band predominantly represented proteins encoded by the Wolbachia genome, including the outer surface protein, Wsp. We further showed that resistance of Wolbachia to streptomycin is associated with a K42R mutation in Wolbachia ribosomal protein S12, and that the pattern of amino acid substitutions in ribosomal protein S12 shows distinct differences in the closely related genera, Wolbachia and Rickettsia.

Upregulation of bone morphogenetic protein GDF-3/Vgr-2 expression in adipose tissue of FABP4/aP2 null mice.

High-fat-fed C57Bl/6J FABP4/aP2 null mice develop obesity but not the related hyperglycemia or hyperinsulinemia characteristic of type II diabetes. FABP4/aP2 protein's function to bind fatty acids in the adipocytes may promote total body energy homeostasis by linking energy depots to the ability to express signaling molecules similar to leptin. To test this hypothesis, proteomic analysis of serum proteins from high-fat-fed wild-type and FABP4/aP2 null mice revealed that the GDF-3/Vgr-2 protein, a bone morphogenetic protein, was upregulated in C57Bl/6J FABP4/aP2 null mice. The increase in serum GDF-3/Vgr-2 protein was correlated with a 27-fold increase in adipose GDF-3/Vgr-2 mRNA. In contrast, leptin expression was unaltered between FABP4/aP2 null and wild-type animals. The expression of GDF-3/Vgr-2 mRNA was not substantially different in adipose tissue of db/db and tb/tb mice compared to wild-type controls. The expression of GDF-3/Vgr-2 mRNA was dependent upon the age and diet of the animals, declining as a function of age in high-fat-fed wild-type animals while increasing in the FABP4/aP2 null strain. These results identify GDF-3/Vgr-2 as an age- and fat-regulated, adipose-derived cytokine suggesting a linkage between adipocyte fatty acid metabolism and the expression of the bone morphogenetic family of differentiation regulators.

Signaling through the hematopoietic cytokine receptors.

Hematopoiesis is regulated through the interaction of a variety of growth factors with specific receptors of the cytokine receptor superfamily. Although lacking catalytic domains, all the receptors couple ligand binding to the rapid induction of protein tyrosine phosphorylation. This is mediated through a novel family of protein tyrosine kinases termed the Janus kinases (Jaks) which associate with the receptors and are activated following ligand binding. Depending upon the cytokine/receptor system, one or more of the four known Jaks (Jak1, Jak2, Jak3, Tyk2) is/are involved. The activated Jaks phosphorylate both themselves and the receptor subunits, creating docking sites for SH2-containing proteins including SHC, which couples receptor engagement to activation of the ras pathway, and HCP, a protein tyrosine phosphatase which negatively affects the response. In addition, the Jaks phosphorylate one or more of a family of signal transducers and activators of transcription (Stats). Phosphorylation of Stats induces their nuclear translocation and DNA-binding activity. Activation of Stats is independent of activation of the ras pathway and represents a novel signaling pathway correlated with mitogenesis.

JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.

Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction.

Interleukin 3 (IL-3) regulates the proliferation and differentiation of hematopoietic cells. Although the IL-3 receptor chains lack kinase catalytic domains, IL-3 induces tyrosine phosphorylation of cellular proteins. To investigate the potential role of the JAK family of protein-tyrosine kinases in IL-3 signal transduction, we have obtained full-length cDNA clones for murine Jak1 and Jak2 protein-tyrosine kinases and prepared antiserum against the predicted proteins. Using antisera against Jak2, we demonstrate that IL-3 stimulation results in the rapid and specific tyrosine phosphorylation of Jak2 and activates its in vitro kinase activity.

Phosphorylation and activation of the DNA binding activity of purified Stat1 by the Janus protein-tyrosine kinases and the epidermal growth factor receptor.

The activation of Janus protein-tyrosine kinases (Jaks) and the subsequent phosphorylation and activation of latent signal transducers and activators of transcription (Stats) are common elements in signal transduction through the cytokine receptor superfamily. To assess the role and specificity of Jaks in Stat activation, we have utilized baculovirus expression systems to produce Stat1 and the Jaks. Co-expression of Stat1 with Tyk2, Jak1, or Jak2 resulted in the specific tyrosine phosphorylation of Stat1 at Tyr701, the residue phosphorylated in mammalian cells stimulated with interferon gamma. Alternatively, Stat1, purified to apparent homogeneity from insect cell extracts, was phosphorylated at Tyr701 in Jak immune complex kinase reactions. Phosphorylation of purified Stat1 was necessary and sufficient for the acquisition of DNA binding activity. The specificity in both systems was indicated by the inability of a Jak2 catalytically inactive mutant (Jak2-Glu882) or the Tec protein-tyrosine kinase to phosphorylate Stat1. However, immune complex-purified epidermal growth factor receptor was capable of phosphorylating purified Stat1 at Tyr701 and activating its DNA binding activity in in vitro reactions.

Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo.

Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane-proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.

Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130.

The interleukin 6 receptor-associated signal transducer, gp130, is shared by receptor complexes for leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and interleukin 11. We show here that JAK2 kinase is rapidly tyrosine phosphorylated in mouse embryonic stem cells whose pluripotentiality is maintained only by gp130-sharing cytokines after stimulation that is known to induce gp130 homodimerization. JAK1 is also tyrosine phosphorylated, but to a lesser extent, under the same conditions. Comparable results are obtained with hemopoietic lineage cells such as myeloid leukemic M1 cells and pro-B-cell line-derived transfectants expressing gp130, the former of which differentiate into macrophages after stimulation of gp130 and the latter of which initiate DNA synthesis. gp130-dimerizing stimulus upregulates kinase activity of JAK2 as revealed by immunocomplex kinase assay. Deletion or point mutation in the membrane-proximal cytoplasmic motifs in gp130 that are conserved in the hemopoietic cytokine receptor family results in the loss of tyrosine phosphorylation of JAK2, which coincides with the lack of signal transducing capability of gp130 mutants.