Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.

Genetic ablation of the src kinase p59fynT exacerbates pulmonary inflammation in an allergic mouse model.

p59fynT is a protein tyrosine kinase in the src family that has been associated with and believed to function in the signaling of many receptors, including the T-cell receptor. A role for the kinase in antigen-driven pulmonary inflammation was examined using mice whose p59fynT gene had been genetically ablated. FynKO mice that were sensitized to ovalbumin exhibited a marked increase in bronchoalveolar lavage eosinophils and cytokines, including interleukin (IL)-4 and IL-5, relative to wild-type mice in response to antigen aerosol exposure. Ovalbumin-stimulated IL-5 production was also increased in cultured splenocytes derived from fynKO mice relative to wild-type mice, whereas interferon-gamma levels were unchanged. Diminished concanavalin A--stimulated IL-4 levels from fynKO splenocytes were consistent with reduced serum immunoglobulin (Ig)E levels observed in sensitized/saline aerosol-challenged animals and may reflect defective natural killer 1.1(+) T cell development. Normalization of IgE levels in sensitized fynKO mice relative to wild-type mice occurred after repeat antigen challenge, which suggests a secondary source of IL-4. Overall, these data demonstrate fyn is a negative regulator of allergic airway inflammation in mice because its absence promotes a shift to a T helper-2 phenotype that may reflect the kinase's role in T-cell receptor signaling.

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

The amino terminus of JAK3 is necessary and sufficient for binding to the common gamma chain and confers the ability to transmit interleukin 2-mediated signals.

JAK3 is a protein tyrosine kinase that specifically associates with the common gamma chain (gammac), a shared subunit of receptors for interleukin (IL) 2, 4, 7, 9, and 15. Patients deficient in either JAK3 or gammac presented with virtually identical forms of severe combined immunodeficiency (SCID), underscoring the importance of the JAK3-gammac interaction. Despite the key roles of JAK3 and gammac in lymphocytic development and function, the molecular basis of this interaction remains poorly understood. In this study, we have characterized the regions of JAK3 involved in gammac association. By developing a number of chimeric JAK3-JAK2 constructs, we show that the binding specificity to gammac can be conferred to JAK2 by transferring the N-terminal domains of JAK3. Moreover, those JAK3-JAK2 chimeras capable of binding gammac were also capable of reconstituting IL-2 signaling as measured by inducible phosphorylation of the chimeric JAK3-JAK2 protein, JAK1, the IL-2 receptor beta chain, and signal transducer and activator of transcription 5A. Subsequent deletion analyses of JAK3 have identified the N-terminal JH7-6 domains as a minimal region sufficient for gammac association. Furthermore, expression of the mutant containing only the JH7-6 domains effectively competed with full-length JAK3 for binding to gammac. We conclude that the JH7-6 domains of JAK3 are necessary and sufficient for gammac association. These studies offer clues toward a broader understanding of JAK-mediated cytokine signaling and may provide a target for the development of novel therapeutic modalities in immunologically mediated diseases.

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity.

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, gammac and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Role of tyrosine kinases in lymphocyte activation: targets for drug intervention.

Recent developments in our understanding of lymphocyte receptor-associated signalling events have offered many new potential targets for modifying antigen and cytokine receptor signalling events in immune-related diseases such as allergy, autoimmunity and transplant rejection. As discussed below, these targets are largely tissue-restricted and are functionally confined to a limited set of receptors. Therefore, it is anticipated that selective inhibitors of these signalling events would offer safe and effective therapies for immunologically-based diseases. First, we review T and B cell antigen receptor signalling as targets for inhibiting lymphocyte responses. Second, targets in lymphocyte cytokine receptor signalling pathways are discussed. Finally, we review strategies for inhibition of receptor signalling.

In vivo augmentation of IFN-gamma with a rIL-12 human/mouse chimera: pleiotropic effects against infectious agents in mice and rats.

A heterodimer containing the mouse 35 kDa and human 40 kDa subunit of IL-12 was expressed in COS cells (cIL-12). Administration of 25-200 U of the cIL-12-COS supernatant to mice twice daily for 2 days augmented spleen cell IFN-gamma production in response to IL-2 and peritoneal macrophage activity (superoxide and nitrites) as compared to animals receiving mock transfected supernatants. cIL-12 also increased levels of IFN-gamma in serum but most dramatically following an LPS injection (50-fold over controls). Animals pretreated with cIL-12 suffered enhanced mortality following challenge with the Gram negative organism E. coli but enhanced survival or clearance following infection with the Gram positive organisms L. monocytogenes and S. aureus. Although daily treatment of mice with cIL-12 following an intranasal influenza A infection elevated levels of IFN-gamma in the bronchioalveolar lavage fluid three-fold over controls, neither prophylactic or therapeutic treatment with the same dose level decreased viral titres in the lung. In addition, no effect was observed in animals infected with encephalomyocarditis virus or respiratory syncytial virus. Therefore, cIL-12 is a potent in vivo augmentor of IFN-gamma production. It has differential effects on infectious disease depending on the invading organism and time of administration; being efficacious for intracellular bacteria but ineffective at the same dose levels against viral diseases.

Purification of the growth-associated protein GAP-43 by reversed phase chromatography: amino acid sequence analysis and cDNA identification.

GAP-43 is a neuronal phosphoprotein. Increased synthesis and axonal transport of GAP-43 has been associated with axon growth, and altered phosphorylation of GAP-43 has been associated with changes in synaptic efficacy. Here we report a rapid and effective procedure employing reverse-phase HPLC for the purification of GAP-43 from rat brain. To characterize the protein purified by this procedure, we generated proteolytic fragments and determined their amino acid sequences. These directly determined sequences, corresponding to 56% of the GAP-43 amino acids, confirm recently reported sequences deduced from the nucleotide sequences of cDNAs. Using oligonucleotide probes constructed according to these amino acid sequences, we identified GAP-43 cDNAs in a library prepared from neonatal rat superior cervical ganglion cells. One of these cDNAs was 1.1 kB in size; it hybridized specifically with a 1.5 kB RNA from brain, but not from liver, and contained the entire coding sequence for GAP-43. This cDNA differed from recently reported cDNAs in its 3' untranslated region.

Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor.

The NGFI-A gene encodes a "zinc-finger" protein that is rapidly induced by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. The complete exon/intron organization and nucleotide sequence of the rat NGFI-A gene have been determined. The gene spans 3789 nucleotides (nt) and is interrupted by a single intron at nt 588. All three zinc-finger DNA-binding domains are contiguously coded for within the 3' exon; this is in contrast to the structure described by others for the Xenopus laevis transcription factor TFIIIA gene. To analyze the transcription of this gene, we have determined the transcription start site and nucleotide sequence of the 5' flanking region. Transfection of PC12 cells with a fragment from the 5' flanking region linked to the chloramphenicol acetyltransferase (CAT) gene revealed that it contains an element which imparts an NGF-inducible phenotype to the normally silent CAT gene. Several regions with homologies to recognizable sequence elements are present in this fragment, including a TATA box at nt -27, serum response elements at nt -84, -106, -370, and -408, a cAMP-responsive element at nt -140, and a transcription factor Sp1-binding site at nt -286. These results establish the genomic structure of this mammalian multifinger protein and demonstrate that an NGF-responsive element lies upstream of the NGFI-A gene.

Tissue-specific phosphorylation of complement receptors CR1 and CR2.

CR1 of neutrophils and monocytes may exist in a resting state, in which it only binds ligand-coated particles, or an activated state, in which it mediates phagocytosis. Because the activated state of CR1 can be induced by the stimulation of protein kinase C with PMA, CR1 was assessed for phosphorylation. Purified human neutrophils, monocytes, eosinophils, tonsilar lymphocytes, SB cells, and erythrocytes were labeled with 32PO4 and incubated with buffer or 100 ng/ml PMA. Membrane proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. CR1, unlike HLA class I heavy chain, was not constitutively phosphorylated by any cell type. PMA induced phosphorylation of CR1 in three phagocytic cell types, but did not induce the phosphorylation of CR3 or FcR. FMLP also induced the phosphorylation of CR1 in neutrophils. In contrast, PMA did not induce phosphorylation of CR1 in tonsilar B lymphocytes, SB cells, or erythrocytes, indicating restriction of this reaction to phagocytic cell types. This may be due to differences in the structure or presentation of the cytoplasmic domain of CR1 in phagocytic vs. nonphagocytic cells. Phosphorylation of CR2, however, did occur in PMA-treated B lymphocytes and SB cells, suggesting that this receptor, rather than CR1, may be involved in regulation of B lymphocyte function.

PMA induces the ligand-independent internalization of CR1 on human neutrophils.

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.

Alpha-fetoprotein inhibits macrophage expression of Ia antigens.

Murine alpha-fetoprotein (AFP) is a major protein in amnionic fluid and perinatal sera. AFP has been postulated to contribute to the immunologic hyporesponsiveness of the fetus and neonate, as well as protecting the fetus from rejection by the mother. We now report that AFP acts in vitro to inhibit macrophage expression of cell surface Ia antigens, the class II glycoproteins of the major histocompatibility gene complex. In culture, macrophages incubated with a T cell lymphokine developed Ia as detected by either immunofluorescence or a cell radioimmunoassay. Both mouse amnionic fluid and AFP inhibited the expression of Ia in a dose-dependent manner. Five preparations of AFP derived from three sources--day-old neonates, amnionic fluid, and a hepatoma cell line--were effective. The concentration of AFP that inhibited by 50% the expression of Ia was about 10(-6) M. Mouse amnionic fluid and AFP did not affect the viability of the macrophage nor was the surface expression of H-2K and C3 receptors affected. The inhibitory effect of AFP did not depend on the presence of T cells in the culture. AFP did not appear to inhibit the direct interaction of lymphokine with macrophages; AFP did inhibit if given days after a pulse of lymphokine. The concentrations of prostaglandins carried by AFP were insignificant and could not explain the inhibitory effects.