Selective modulation of chemokinesis, degranulation, and apoptosis in eosinophils through the PGD2 receptors CRTH2 and DP.

BACKGROUND: PGD(2) is the major prostanoid released by mast cells during an allergic response. Its role in the allergic response, however, remains unclear. OBJECTIVE: Because the accumulation of eosinophils is a feature of allergic reactions, we investigated the role of PGD(2) in the modulation of eosinophil function. METHODS: Circulating human eosinophils were isolated and challenged with PGD(2). The effects of PGD(2) on various eosinophil functions were then analyzed. RESULTS: PGD(2) binds with high affinity preferentially to 2 receptors, DP and chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2). We show that both DP and CRTH2 are detectable on circulating eosinophils. We demonstrate that PGD(2) (1-10 nmol/L) induces a rapid change in human eosinophil morphology and an increase in chemokinesis and promotes eosinophil degranulation. These effects are induced by the CRTH2-selective agonist 13-14-dihydro-15-keto-PGD(2) (DK-PGD(2)) but not by the DP-selective agonist BW245C. These results suggest a role for CRTH2 in the modulation of eosinophil movement and in triggering the release of cytotoxic proteins. Finally, we demonstrate that BW245C, but not DK-PGD(2), can delay the onset of apoptosis in cultured eosinophils, presumably through interaction with DP. CONCLUSION: These data support the hypothesis that PGD(2) controls eosinophil functions through 2 pharmacologically distinct receptors with independent functions. Blockade of PGD(2)-mediated effects on human eosinophils may reduce the damage caused by these cells during an allergic response, but inhibition of both receptors may be required.

Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain.

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.

Interactions of CD45-associated protein with the antigen receptor signaling machinery in T-lymphocytes.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.

Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation.

The amyloid-beta precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-beta (A beta) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimer's disease brains and colocalization of its APP cleavage product with A beta in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for A beta peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimer's disease.

Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide.

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.

Reconstitution of interactions between protein-tyrosine phosphatase CD45 and tyrosine-protein kinase p56(lck) in nonlymphoid cells.

To further understand the functional interactions between CD45 and p56(lck) in T-cells, we stably reconstituted their expression in a nonlymphoid system. The results of our analyses demonstrated that CD45 could dephosphorylate tyrosine 505 of p56(lck) in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56(lck) interfered with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56(lck). The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells was also greatly influenced by the catalytic activity of p56(lck). Indeed, whereas CD45 provoked dephosphorylation of kinase-defective Lck molecules in this system, it failed to stably dephosphorylate kinase-active p56(lck) polypeptides. Finally, our studies showed that CD45 was also able to inhibit the oncogenic potential of a constitutively activated version of p56(lck) in NIH 3T3 cells. This effect did not require the Lck unique domain and apparently resulted from selective dephosphorylation of substrates of activated p56(lck) in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56(lck) in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56(lck)-mediated functions in vivo.

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

Ntk: a Csk-related protein-tyrosine kinase expressed in brain and T lymphocytes.

The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this study, we characterize Ntk, a protein-tyrosine kinase bearing striking similarities to p50csk. Like p50csk, Ntk possesses Src homology 3 and Src homology 2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in members of the Src family. Expression of ntk transcripts was maximal in brain, and was observed at significant levels in thymus and spleen. ntk RNA levels were dramatically reduced upon mitogenic stimulation of normal T lymphocytes and were minimal in transformed T-cell populations. Firm evidence that Ntk is a Csk-related enzyme was provided by the observation that it phosphorylated a Src-related polypeptide on the inhibitory carboxyl-terminal tyrosine. These findings indicate that Ntk is a Csk-related enzyme that may play an inhibitory role in the control of T-cell proliferation.

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

Interactions of the SH2 domain of lymphocyte-specific tyrosine protein kinase p56lck with phosphotyrosine-containing proteins.

We have previously demonstrated that the non-catalytic Src homology 2 (SH2) domain is required for both positive and negative regulation of the catalytic function of the lymphocyte-specific tyrosine protein kinase p56lck. Indeed, the ability of activated p56lck molecules (tyrosine 505 to phenylalanine 505 mutants) to enhance T-cell receptor (TCR)-induced tyrosine protein phosphorylation is dramatically reduced by deletion of the SH2 domain. Paradoxically, removal of the SH2 sequence also results in constitutive elevation of the catalytic function of wild-type Lck polypeptides, rendering them capable of oncogenic transformation of rodent fibroblasts. As SH2 sequences can mediate binding to phosphotyrosine-containing peptides, the ability of the Lck SH2 domain to interact with tyrosine-phosphorylated proteins was tested. We found that the SH2 sequence of p56lck can bind several of the TCR-regulated tyrosine phosphorylation substrates in vitro. One of the substrates, an 80-kilodalton (kDa) phosphoprotein (p80) showed the tightest binding to the SH2 domain of Lck. Additionally, it was observed that the SH2 domain of Lck can bind a synthetic peptide containing the phosphorylated carboxy-terminal tyrosine 505 of p56lck. Indirect evidence indicating that the SH2 region interacts with the tyrosine-phosphorylated carboxy terminus of Lck in vivo was also obtained. As deletion of the SH2 domain or mutation of tyrosine 505 results in p56lck activation in vivo, it is conceivable that interactions between these two regions impose a conformation that is unfavorable to phosphorylation of intracellular substrates. Collectively, these findings suggest that the SH2 domain modulates the catalytic function of Lck through complex interactions with phosphotyrosine-containing proteins.

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.

The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression.

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.