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D B Straus

Publications and source records attributed to D B Straus.

At least 19 recordsLinked to original sources
PubMed2001

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.

B-Lymphocytes↗
PubMed2000

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Alkyl and Aryl Transferases↗
PubMed2000

CD28 and the tyrosine kinase lck stimulate mitogen-activated protein kinase activity in T cells via inhibition of the small G protein Rap1.

Proliferation of T cells via activation of the T-cell receptor (TCR) requires concurrent engagement of accessory costimulatory molecules to achieve full activation. The best-studied costimulatory molecule, CD28, achieves these effects, in part, by augmenting signals from the TCR to the mitogen-activated protein (MAP) kinase cascade. We show here that TCR-mediated stimulation of MAP kinase extracellular-signal-regulated kinases (ERKs) is limited by activation of the Ras antagonist Rap1. CD28 increases ERK signaling by blocking Rap1 action. CD28 inhibits Rap1 activation because it selectively stimulates an extrinsic Rap1 GTPase activity. The ability of CD28 to stimulate Rap1 GTPase activity was dependent on the tyrosine kinase Lck. Our results suggest that CD28-mediated Rap1 GTPase-activating protein activation can help explain the augmentation of ERKs during CD28 costimulation.

Animals↗
PubMed2000

Differential T-cell antigen receptor signaling mediated by the Src family kinases Lck and Fyn.

Src family tyrosine kinases play a key role in T-cell antigen receptor (TCR) signaling. They are responsible for the initial tyrosine phosphorylation of the receptor, leading to the recruitment of the ZAP-70 tyrosine kinase, as well as the subsequent phosphorylation and activation of ZAP-70. Molecular and genetic evidence indicates that both the Fyn and Lck members of the Src family can participate in TCR signal transduction; however, it is unclear to what extent they utilize the same signal transduction pathways and activate the same downstream events. We have addressed this issue by examining the ability of Fyn to mediate TCR signal transduction in an Lck-deficient T-cell line (JCaM1). Fyn was able to induce tyrosine phosphorylation of the TCR and recruitment of the ZAP-70 kinase, but the pattern of TCR phosphorylation was altered and activation of ZAP-70 was defective. Despite this, the SLP-76 adapter protein was inducibly tyrosine phosphorylated, and both the Ras-mitogen-activated protein kinase and the phosphatidylinositol 4, 5-biphosphate signaling pathways were activated. TCR stimulation of JCaM1/Fyn cells induced the expression of the CD69 activation marker and inhibited cell growth, but NFAT activation and the production of interleukin-2 were markedly reduced. These results indicate that Fyn mediates an alternative form of TCR signaling which is independent of ZAP-70 activation and generates a distinct cellular phenotype. Furthermore, these findings imply that the outcome of TCR signal transduction may be determined by which Src family kinase is used to initiate signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Alanine↗
PubMed1999

The lck SH3 domain is required for activation of the mitogen-activated protein kinase pathway but not the initiation of T-cell antigen receptor signaling.

Initiation of T-cell antigen receptor (TCR) signaling is dependent upon the activity of protein tyrosine kinases. The Src family kinase Lck is required for the initial events in TCR signaling, such as the phosphorylation of the TCR complex and the activation of ZAP-70, but little is known of its role in downstream signaling. Expression of a mutated form of Lck lacking SH3 domain function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a requirement for Lck beyond the initiation of TCR signaling. In cells expressing LckW97A, stimulation of the TCR failed to activate the mitogen-activated protein kinase (MAPK) pathway, despite normal TCR zeta chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation. Activation of extracellular signal-regulated kinase (ERK) and MAPK kinase (MEK), as well as the induction of CD69 expression, was greatly impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of phospholipase Cgamma1 (PLCgamma1) and corresponding elevations in intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells expressing LckW97A exhibit a selective defect in the activation of the MAPK pathway. These results demonstrate that Lck has a role in the activation of signaling pathways beyond the initiation of TCR signaling and suggest that the MAPK pathway may be selectively controlled by regulating the function of Lck.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

SH2 domain function is essential for the role of the Lck tyrosine kinase in T cell receptor signal transduction.

Tyrosine kinase activity is required for signal transduction through the T cell antigen receptor (TCR). The Src family tyrosine kinase Lck appears to play a key role in the initiation of TCR signaling events. We have investigated the role of the phosphotyrosine-binding Src homology-2 (SH2), domain of Lck in TCR signaling. Lck containing a mutation in the phosphotyrosine binding pocket of the SH2 domain was expressed in an Lck-deficient cell line. We found that, in contrast to wild-type Lck, the SH2 domain mutant was unable to restore even the earliest TCR-mediated signaling events. To investigate the role of the Lck SH2 domain, we examined the association of tyrosine phosphoproteins with Lck. The predominant associated phosphoprotein was the ZAP-70 tyrosine kinase, which has also been implicated in the initiation of TCR signaling. In addition, the zeta subunit of the T cell receptor was found to weakly associate with Lck. Further analysis indicated that the SH2 domain of Lck can directly recognize both ZAP-70 and zeta in immunoprecipitates from TCR-stimulated cells. Our findings demonstrate that the SH2 domain of Lck is essential for the initiation of signaling events following TCR stimulation probably as a result of its ability to mediate an interaction between Lck and the ZAP-70 tyrosine kinase and/or the zeta subunit of the T cell receptor.

Calcium↗
PubMed1993

The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation.

Recent work indicates that signaling events resulting from stimulation of the T cell antigen receptor (TCR) can be initiated by the CD3 complex (gamma, delta, epsilon) as well as the zeta chains of the receptor. To help characterize the signaling function of CD3 we examined its associated tyrosine kinase activity since induction of tyrosine phosphorylation is one of the earliest signaling events. Our results indicate that at least two kinases, lck and ZAP-70, contribute to the CD3-associated kinase activity. A likely target of this activity is the CD3 complex itself since we observed that TCR stimulation resulted in rapid tyrosine phosphorylation of the CD3 epsilon and delta chains. To examine the function of the CD3 epsilon chain in particular, we constructed a chimera that fused the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of CD3 epsilon. This chimera demonstrated that CD3 epsilon was independently capable of associating with proteins having tyrosine kinase activity, including ZAP-70. Our results show that the kinase activity that associates with the CD3 complex has characteristics that are quite similar to the previously characterized zeta-associated kinase activity. This finding suggests that both these components of the TCR initiate signaling events using a common mechanism. However, differences in their signaling function could result from recognition of distinct substrates.

Antibodies, Monoclonal↗
PubMed1992

Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor.

Signaling through the T cell antigen receptor (TCR) results both in rapid increases in tyrosine phosphorylation on a number of proteins and in the activation of the phosphatidylinositol pathway. It is not clear how stimulation of the TCR leads to these signaling events. Mutants of the Jurkat T cell line have been previously isolated that fail to show increases in calcium following receptor stimulation. Analysis of one of these mutants, JCaM1, which is defective in the induction of tyrosine phosphorylation, revealed a defect in the expression of functional lck tyrosine kinase. The lack of lck activity was caused in part by a splicing defect. Expression of the lck cDNA in JCaM1 restores the ability of the cell to respond to TCR stimulation. These results indicate that lck is required for normal signal transduction through the TCR.

Cell Line↗
PubMed1989

The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli.

The expression of heat shock genes in Escherichia coli is controlled by the action of an alternate sigma-factor of RNA polymerase, sigma 32, which directs core RNA polymerase to recognize the promoters for heat shock genes. After a shift from 30 degrees C to 42 degrees C, both the level of sigma 32 and transcription initiation at heat shock promoters transiently increase, indicating that heat shock gene expression is regulated by changes in the concentration of sigma 32. Here, we report that heat shock gene expression is regulated by changes in the activity of sigma 32 under some conditions. Our results show that the transient repression of heat shock protein synthesis, which follows a shift down from 42 degrees C to 30 degrees, occurs as a result of decreased transcription initiation at heat shock promoters, but this repression is accompanied by only a small decrease in the level of sigma 32. In addition, the induction of heat shock proteins following overproduction of sigma 32 from a multicopy plasmid is only transient, despite the fact that the level of sigma 32 remains elevated. Constitutive overproduction of sigma 32 also fails to cause a proportionate increase in heat shock gene transcription. These three examples suggest that the activity of sigma 32 is reduced under conditions of excess heat shock gene expression.

Chloramphenicol↗
PubMed1988

Escherichia coli heat shock gene mutants are defective in proteolysis.

Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown. The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides. We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase. Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis. Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay. The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation. These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides.

Adenosine Triphosphate↗
PubMed1987

Induction of the heat shock response of E. coli through stabilization of sigma 32 by the phage lambda cIII protein.

The cIII protein of phage lambda favors the lysogenic response to infection by inhibiting the degradation of the lambda cII protein, which exerts the primary control on the developmental decision for lysis or lysogeny. To study the mechanism and scope of cIII-mediated regulation, we have used plasmid systems to examine the specific effect of cIII overproduction on the growth of Escherichia coli and the synthesis of bacterial proteins. We have found that maximal production of cIII prolongs the heat-induced synthesis of E. coli heat shock proteins and provokes elevated production of heat shock proteins even at low temperature. The overproduction of heat shock proteins is correlated with a rapid inhibition of cell growth, as judged by measurements of optical density. We suggest that an overactive heat shock response inhibits bacterial growth, either because excessive production of one or more of the proteins is highly deleterious or because only heat shock promoters are transcribed efficiently. To examine the effect of cIII on sigma 32, the specificity factor for the heat shock response, we have studied the stability of sigma 32 in cells carrying both cIII- and sigma 32-producing plasmids; the half-life of sigma 32 is increased fourfold in the presence of cIII. We conclude that overproduction of cIII provokes the heat shock response by increasing the steady-state level of active sigma 32. These studies also support the concept that the rate of expression of heat shock proteins is directly correlated with the amount of active sigma 32 and that regulation of the stability of sigma 32 may be an important factor for control of the heat shock response.

Bacteriophage lambda↗
PubMed1987

Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli.

The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response. We present experiments indicating a direct role for sigma 32 in controlling the heat shock response. Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32. Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32. These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.

Escherichia coli↗
PubMed1984

Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase.

The rpoD gene encoding the sigma subunit of E. coli RNA polymerase is cotranscribed with rpsU and dnaG, encoding ribosomal protein S21 and DNA primase, respectively. After temperature upshift, a heat shock promoter (Phs) located within dnaG is transiently induced, causing increased transcription of rpoD. The extent of induction is sufficient to account for the heat shock response of sigma synthesis. The initiation site of this promoter was located about 360 bp upstream of rpoD by promoter cloning and S1 nuclease mapping. Plasmid deletions generated with Bal 31 nuclease show that the DNA sequence CTGCCACCC in the -44 to -36 region of this promoter is necessary for its heat shock activity. Heat induction of transcription from Phs is under the control of HtpR, a positive regulator of the heat shock response.

Base Sequence↗