Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src.

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through adhesion receptors known as integrins. Here we show that integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alpha v but not with beta 1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor that acts at the integrin-cytoskeleton interface to regulate cell spreading and migration.

Tec family kinases modulate thresholds for thymocyte development and selection.

Tec family kinases are implicated in T cell receptor (TCR) signaling, and combined mutation of inducible T cell kinase (Itk) and resting lymphocyte kinase (Rlk)/Txk in mice dramatically impairs mature T cell function. Nonetheless, mutation of these kinases still permits T cell development. While itk(-)(/)- mice exhibit mild reductions in T cells with decreased CD4/CD8 cell ratios, rlk(-)(/)-itk(-)(/)- mice have improved total T cell numbers yet maintain decreased CD4/CD8 ratios. Using TCR transgenics and an in vitro thymocyte deletion model, we demonstrate that mutation of Tec kinases causes graded defects in thymocyte selection, leading to a switch from negative to positive selection in rlk(-)(/)-itk(-)(/)- animals. The reduction in both positive and negative selection and decreased CD4/CD8 ratios correlates with decreased biochemical parameters of TCR signaling, specifically defects in capacitive Ca(2+) influx and activation of the mitogen-activated kinases extracellular signal-regulated kinase 1 and 2. Thus, Tec kinases influence cell fate determination by modulating TCR signaling, leading to altered thresholds for thymocyte selection. These results provide support for a quantitative model for thymic development and provide evidence that defects in negative selection can substantially alter thymic cellularity.

Sublethal gamma-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor beta rearrangement in recombinase activation gene 2-/- mice.

DNA recombination of the immunoglobulin (Ig) or T cell receptor (TCR) gene loci is an essential step in the production of lymphocytes bearing antigen-specific receptors. Mice that lack the ability to rearrange their Ig and TCR gene loci are devoid of mature B and T cells. Complete rearrangement and expression of the TCR-beta chain has been suggested to allow immature thymocytes to switch from the CD4-/CD8- to the CD4+/CD8+ stage of thymic development. Thus, thymocytes from severe combined immune deficient (SCID) mice or mice deficient in recombinase activation genes (RAG), which do not undergo proper DNA rearrangement, are arrested at the early CD4-/CD8- stage of development. B cell precursors in SCID or RAG mice do not progress from the B220+/sIgM-/heat stable antigen (HSA)+/CD43+ to the B220+/sIgM-/HSA+/CD43- stage. In an attempt to reconstitute RAG-2-/- mice with bone marrow- or fetal liver-derived progenitor cells, we subjected these mice to sublethal doses of gamma-radiation. It is surprising that in the absence of donor cells, irradiated RAG-2-/- mice revealed a dramatic change in their lymphoid phenotype. 14 d after irradiation, the majority of thymocytes had advanced to the CD4+/CD8+ stage of T cell development and a small number of bone marrow precursors had progressed to the CD43-, HSAhi stage of B cell development. Analysis of the resulting CD4+/CD8+ thymocytes revealed no surface expression of the TCR/CD3 complex and no V-D-J rearrangement of the TCR-beta gene locus. Our findings provide evidence for a novel pathway that allows the transition of thymocytes from the CD4-/CD8- to the CD4+/CD8+ stage and that does not appear to require TCR-beta chain rearrangement.

Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells.

A substitution mutation has been introduced into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene, and these cells have been used to generate chimeric mice. It is shown that the c-abl mutation was transmitted to progeny by several male chimeras. This work demonstrates the feasibility of germ-line transmission of a mutation introduced into a nonselectable autosomal gene by homologous recombination.

Requirement for Tec kinases Rlk and Itk in T cell receptor signaling and immunity.

T cell receptor (TCR) signaling requires activation of Zap-70 and Src family tyrosine kinases, but requirements for other tyrosine kinases are less clear. Combined deletion in mice of two Tec kinases, Rlk and Itk, caused marked defects in TCR responses including proliferation, cytokine production, and apoptosis in vitro and adaptive immune responses to Toxoplasma gondii in vivo. Molecular events immediately downstream from the TCR were intact in rlk-/-itk-/- cells, but intermediate events including inositol trisphosphate production, calcium mobilization, and mitogen-activated protein kinase activation were impaired, establishing Tec kinases as critical regulators of TCR signaling required for phospholipase C-gamma activation.

Tec family kinases in lymphocyte signaling and function.

The Tec kinases are required for full Ca(2+) mobilization in lymphocytes. Recent data suggest that this process occurs via a multiprotein complex that includes LAT and SLP-76 in T cells and BLNK/SLP-65 in B cells. Mutational analyses have revealed critical roles for Tec kinases in lymphocyte development and function.

Tec kinases: modulators of lymphocyte signaling and development.

The Tec kinases are implicated as important components of the antigen receptor signaling required for proper lymphocyte activation and development. Recent data suggest that these kinases contribute to multiprotein complexes containing LAT and SLP-76 in T cells, and BLNK/SLP-65 in B cells, which are required for activation of PLC-gamma and downstream pathways.

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

rlk/TXK encodes two forms of a novel cysteine string tyrosine kinase activated by Src family kinases.

Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.

CD84 mediates CLL-microenvironment interactions.

Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

The many faces of Src: multiple functions of a prototypical tyrosine kinase.

c-src was first isolated as the normal cellular homologue of v-src, the transforming gene of Rous Sarcoma virus (Stehelin et al., 1976). As the first proto-oncogene described and one of the first molecules demonstrated to have tyrosine kinase activity, Src has provided a prototype for understanding signal transduction involving tyrosine phosphorylation. Comparison between c-src and activated or transforming mutants of Src including v-src, combined with recent data on the structure of Src family kinases has provided new insight into their regulation. In this review, I will discuss the function of the various domains of Src in light of these mutational and structural studies.

Immune responses in mice deficient in Ly-GDI, a lymphoid-specific regulator of Rho GTPases.

Ly-GDI (lymphoid-specific guanosine diphosphate (GDP) dissociation inhibitor), also called D4-GDI, is preferentially expressed in hematopoietic tissues including bone marrow, thymus, spleen and lymph nodes. It binds to the small guanosine triphosphate (GTP)-binding protein Rho and inhibits GDP dissociation from Rho proteins. To explore the function of Ly-GDI in lymphocytes, we have generated Ly-GDI-deficient mice by gene targeting. These mice showed no striking abnormalities of lymphoid development or thymocyte selection. The mice also exhibited, for the most part, normal immune responses including lymphocyte proliferation, IL-2 production, cytotoxic T lymphocyte activity, antibody production, antigen processing and presentation, immune cell aggregation and migration, and protection against an intracellular protozoan. However, Ly-GDI-deficient mice exhibited deregulated T and B cell interactions after in vitro cultivation of mixed lymphocyte populations in concanavalin A (Con A) leading to overexpansion of B lymphocytes. Further studies revealed that Ly-GDI deficiency decreased IL-2 withdrawal apoptosis of lymph node cells while dexamethasone- and T cell receptor-induced apoptosis remained intact. These data implicate the regulation of the Rho GTPase by Ly-GDI in lymphocyte survival and responsiveness, but suggest that these functions may be partially complemented by other Rho regulatory proteins when the Ly-GDI protein is deficient.

Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk.

BACKGROUND: The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. RESULTS: We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. CONCLUSIONS: Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.

Cloning and characterization of an inversion breakpoint at 6q23.3 suggests a role for Map7 in sacral dysgenesis.

Here we report on a male patient with sacral dysgenesis (SD) and constitutional pericentric inversion of chromosome 6 (p11.2;q23.3). SD is a heterogeneous group of congenital anomalies with complex genetic etiology. Previously, a patient with sacral abnormalities and an interstitial deletion of 6q23-->q25 region has been described. We speculated that a susceptibility gene for SD lies in 6q23.3 region (disrupted in both patients), and therefore, cloning of the breakpoint in our patient would lead to the identification of the disrupted gene. We performed FISH analysis followed by Southern blot analysis and inverse PCR to clone the breakpoint. The 6p11.2 breakpoint mapped very close to the centromere, and the 6q23.3 breakpoint localized in the ninth intron of the MAP7 gene. We then evaluated the involvement of MAP7 in SD by further screening of the gene in several patients with a similar phenotype. Two nucleotide changes causing Ile257Asn and Glu571Ala substitutions in the protein, both affecting amino acid residues conserved in the mouse homolog, were identified in two patients. Both changes are either very rare polymorphisms or true mutations, since they were not detected in 167 normal individuals nor found in the SNP database. Therefore, our study suggests MAP7 as a candidate gene for SD. However, we were unable to detect any sacral defects in the MAP7 knockout mice.

Clinical reproducibility of dual energy x-ray absorptiometry.

Dual energy x-ray absorptiometry is a technique advocated for the measurement of bone mass throughout the skeleton, and recently it has been used to measure changes in periprosthetic bone mass after joint replacement. The accuracy and precision of the method in clinical patient populations have not been firmly established. This study sought to establish the short-term reproducibility of measurements made with dual energy x-ray absorptiometry of multiple sites in a large sample of elderly patients with rheumatic disease. Reproducibility was assessed in the lumbar spine and in three femoral sites in 69 patients participating in a longitudinal clinical trial. In each patient, absorptiometry was performed twice in the same day at as many as five time points over a 2-year period. The mean (+/- SD) baseline bone density was 0.783 +/- 0.128 g/cm2 for the femoral neck and 1.015 +/- 0.218 g/cm2 for the lumbar spine. The correlations between the duplicate baseline measurements of the spine were excellent (r = 0.9936, p < 0.001) and were stable over the 2-year period; the mean difference between the duplicate baseline measurements was 1.82 +/- 1.54% and the mean coefficient of variation was 1.29%. Measurements in the femur were much less precise; these values were 3.61 +/- 3.14% and 2.55% in the femoral neck, 3.66 +/- 4.35% and 2.59% in the greater trochanter, and 5.28 +/- 5.61% and 3.73% in Ward's triangle. This study evaluated the short-term reproducibility of dual energy x-ray absorptiometry in a clinical population.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence.

Linear retroviral DNA, the major precursor to the integrated provirus of the murine leukemia viruses, contains a mixture of two structures at its ends: some termini are full-length and blunt, and some have recessed 3' strands. A temporal study of the end structures showed that the proportion of the DNA with recessed ends increases during the course of infection, and suggests that the blunt ends are precursors to the recessed ends. We have examined the DNA structures of the ends of retroviral mutants defective in the integration (IN) function. The results show that the formation of the recessed ends requires the presence of IN. Finally, we have analyzed the structures at the ends of mutant genomes with alterations in the terminal DNA sequence. The exact position of the recessed 3' end can be recessed one, two, or four nucleotides relative to the 5' end. In all cases the position of the recessed 3' end correlates perfectly with, and thus presumably determines, the site of joining to the target DNA.

Point mutations in the P30 domain of the gag gene of Moloney murine leukemia virus.

A series of point mutations in the P30 domain of the Moloney murine leukemia virus gag gene was generated by bisulfite treatment of heteroduplex DNAs containing a single-stranded region in the gag gene. One virus bearing such a mutation exhibited a coordinate defect in gag and pol function, and was similar to previously described deletion mutants with alterations in this gene. One mutant virus displayed a different phenotype: it could assemble virion particles and provide pol function, but the particles were defective in the early stages of infection. The continued concordance of the mutants' failure or ability to both assemble virions and provide pol lends further support to the proposal that similar parts of the gag gene are required for these two processes.