An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.

Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.

Long-term potentiation increases tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit 2B in rat dentate gyrus in vivo.

Long-term potentiation (LTP) is a form of synaptic memory that may subserve developmental and behavioral plasticity. An intensively investigated form of LTP is dependent upon N-methyl-D-aspartate (NMDA) receptors and can be elicited in the dentate gyrus and hippocampal CA1. Induction of this type of LTP is triggered by influx of Ca2+ through activated NMDA receptors, but the downstream mechanisms of induction, and even more so of LTP maintenance, remain controversial. It has been reported that the function of NMDA receptor channel can be regulated by protein tyrosine kinases and protein phosphatases and that inhibition of protein tyrosine kinases impairs induction of LTP. Herein we report that LTP in the dentate gyrus is specifically correlated with tyrosine phosphorylation of the NMDA receptor subunit 2B in an NMDA receptor-dependent manner. The effect is observed with a delay of several minutes after LTP induction and persists in vivo for several hours. The potential relevance of this post-translational modification to mechanisms of LTP and circuit plasticity is discussed.

Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.

Clusters of charged residues in protein three-dimensional structures.

Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.

The stress response to ionizing radiation involoves c-Abl-dependent phosphorylation of SHPTP1.

c-Abl is a nonreceptor tyrosine kinase that is activated by certain DNA-damaging agents. The present studies demonstrate that nuclear c-Abl binds constitutively to the protein tyrosine phosphatase SHPTP1. Treatment with ionizing radiation is associated with c-Abl-dependent tyrosine phosphorylation of SHPTP1. The results demonstrate that the SH3 domain of c-Abl interacts with a WPDHGVPSEP motif (residues 417-426) in the catalytic domain of SHPTP1 and that c-Abl phosphorylates C terminal Y536 and Y564 sites. The functional significance of the c-Abl-SHPTP1 interaction is supported by the demonstration that, like c-Abl, SHPTP1 regulates the induction of Jun kinase activity following DNA damage. These findings indicate that SHPTP1 is involved in the response to genotoxic stress through a c-Abl-dependent mechanism.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Structural analysis of p185c-neu and epidermal growth factor receptor tyrosine kinases: oligomerization of kinase domains.

The epidermal growth factor receptor (EGFR) and p185c-neu proteins associate as dimers to create an efficient signaling assembly. Overexpression of these receptors together enhances their intrinsic kinase activity and concomitantly results in oncogenic cellular transformation. The ectodomain is able to stabilize the dimer, whereas the kinase domain mediates biological activity. Here we analyze potential interactions of the cytoplasmic kinase domains of the EGFR and p185c-neu tyrosine kinases by homology molecular modeling. This analysis indicates that kinase domains can associate as dimers and, based on intermolecular interaction calculations, that heterodimer formation is favored over homodimers. The study also predicts that the self-autophosphorylation sites located within the kinase domains are not likely to interfere with tyrosine kinase activity, but may regulate the selection of substrates, thereby modulating signal transduction. In addition, the models suggest that the kinase domains of EGFR and p185c-neu can undergo higher order aggregation such as the formation of tetramers. Formation of tetrameric complexes may explain some of the experimentally observed features of their ligand affinity and hetero-receptor internalization.

IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.