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At least 883 records · Page 49Linked to original sources
PubMed2005

Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: regulatory role of serine 1223.

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed2006

The nuclear hormone receptor farnesoid X receptor (FXR) is activated by androsterone.

Farnesoid X receptor (FXR) uses bile acids as endogenous ligands. Here, we demonstrate that androsterone, a metabolic product of testosterone, is also an FXR ligand. Treatment of castrated male mice with androsterone induced expression of the FXR target gene small heterodimer partner (SHP). In mouse AML-12 hepatocytes, chenodeoxycholic acid (CDCA) or androsterone induced SHP expression with a similar kinetic pattern. The FXR antagonist guggulsterone blocked the induction of SHP by androsterone in AML-12 cells. Nuclear magnetic resonance spectroscopy demonstrated the direct binding of androsterone to purified human FXR (hFXR) ligand-binding domain (LBD) protein, resulting in the recruitment of steroid receptor coactivator protein-1 (SRC-1) coactivator peptide. In HEK293 cells, androsterone activated gal4-mouse FXR-LBD and gal4-hFXR-LBD fusion proteins, although in contrast to CDCA, androsterone activation was significantly greater for the mouse FXR-LBD than for the hFXR-LBD. Site-directed mutagenesis of the hFXR-LBD defined amino acids Asn354 and Ser345 as critical for differential species sensitivity to CDCA and androsterone, respectively. Crystal structure studies suggest that the orientation of the steroid nucleus of bile acids within the binding pocket of FXR is reversed from all other nuclear hormone receptors. In support of this model, we show here that mutations M265I or R331H, residues predicted by crystal structure to interact with the carboxylic acid tail of CDCA but not with androsterone, altered CDCA activation but had no effect on androsterone activation. Activation of FXR by androsterone may provide an additional means for physiological or pharmacological modulation of FXR.

Amino Acid Sequence↗
PubMed1983

Differentiation of pseudo- and idiopathic hypoparathyroidism by measuring urinary calcitonin.

Calcitonin (CT) levels were determined in urine specimens from normal subjects and patients with pseudohypoparathyroidism type I (PHP), idiopathic hypoparathyroidism (IHP), pseudopseudohypoparathyroidism (PPHP), and surgical hypoparathyroidism (SHP). Urinary CT was measured by RIA after extraction of urine by gel chromatography on a 0.8 X 20-cm column of Bio-Gel P-2. The urinary CT level ranged from 420-1000 pg/mg creatinine (Cr; mean +/- SD, 631 +/- 229) in PHP (n = 6), from 50-270 (131 +/- 92) in IHP (n = 6), and from 35-93 (66 +/- 27) in SHP (n = 6), and was 185 pg/mg Cr in one patient with PPHP. The mean value in PHP was significantly (P less than 0.001) higher, and that in SHP was significantly (P less than 0.05) lower, than those in the age-matched normal subjects. In each patient with PHP and IHP before and after treatment with 1 alpha-hydroxycholecalciferol, urinary CT levels fluctuated between 700-1370 pg/mg Cr in PHP and from 43-195 pg/mg Cr in IHP. Increased urinary excretion of CT in patients with PHP suggests that CT secretion may be enhanced in this disease. The biological role of CT in PHP remains to be clarified.

Adolescent↗
PubMed2002

Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells.

In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85alpha subunit of PI3K, but led to a significant reduction of insulin-induced p85alpha phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.

Animals↗
PubMed2006

Functional analysis of SIRPalpha in the growth cone.

The 'signal regulatory protein' SIRPalpha is an Ig superfamily, transmembrane glycoprotein with a pair of cytoplasmic domains that can bind the phosphatase SHP-2 when phosphorylated on tyrosine. SIRPalpha is prominent in growth cones of rat cortical neurons and located, together with the tetraspanin CD81, in the growth cone periphery. SIRPalpha is dynamically associated with Triton-X-100-sensitive, but Brij-98-resistant, lipid microdomains, which also contain CD81. Challenge of growth cones with the integrin-binding extracellular-matrix (ECM) protein, laminin, or with the growth factors, IGF-1 or BDNF, increases SIRPalpha phosphorylation and SHP-2 binding rapidly and transiently, via Src family kinase activation; phosphorylated SIRPalpha dissociates from the lipid microdomains. A cytoplasmic tail fragment of SIRPalpha (cSIRPalpha), when expressed in primary cortical neurons, also is phosphorylated and binds SHP-2. Expression of wild-type cSIRPalpha, but not of a phosphorylation-deficient mutant, substantially decreases IGF-1-stimulated axonal growth on laminin. On poly-D-lysine and in control conditions, axonal growth is slower than on laminin, but there is no further reduction in growth rate induced by the expression of cSIRPalpha. Thus, the effect of cSIRPalpha on axon growth is dependent upon integrin activation by laminin. These results suggest that SIRPalpha functions in the modulation of axonal growth by ECM molecules, such as laminin.

Animals↗
PubMed2002

Mast cell regulation via paired immunoglobulin-like receptor PIR-B.

Activating (PIR-A) and inhibitory (PIR-B) isoforms of the paired immunoglobulin (Ig)-like receptor family have been evaluated for their modulating potential in mast cell responses to IgE antibody and mast/stem cell growth factor (SCF). Mast cells produce PIR-A and PIR-B, but PIR-B was found to be predominantly expressed on the cell surface, where it was constitutively tyrosine phosphorylated and associated with SHP-1 tyrosine phosphatase. Efficient coligation of PIR-B with FcepsilonRI inhibited IgE-induced mast cell activation and serotonin release. PIR-B and c-kit (or mast/SCF receptor) coligation also inhibited SCF-induced mast cell responses. The PIR-B inhibitory activity was unimpaired in SHP-1-deficient mast cells, perhaps because of non-SHP-1-binding tyrosine-based inhibitory motif in the cytoplasmic tail of PIR-B. This analysis suggests that PIR-B may serve to control mast cell activity.

Animals↗
PubMed2006

The role of Helicobacter pylori CagA in gastric carcinogenesis.

Gastric carcinoma is the second most common cause of cancer-related deaths in the world, accounting for more than 400,000 deaths each year. Infection with cagA-positive Helicobacter pylori plays a pivotal role in the development of gastric adenocarcinoma. The cagA gene product CagA is directly delivered into gastric epithelial cells via the type IV secretion system. Following membrane localization and subsequent tyrosine phosphorylation by Src family kinases, CagA interacts with a variety of host cell proteins that are involved in the regulation of cell growth and motility in both phosphorylation-dependent and -independent manners. Of special interest is the interaction of CagA with the SH2 domain-containing tyrosine phosphatase SHP-2, gain-of-function mutations of which have recently been found in human malignancies. CagA binds to and activates SHP-2 in a tyrosine phosphorylation-dependent manner, thereby provoking abnormal activation of Erk MAP kinase while inducing elevated cell motility. Perturbation of SHP-2 and other signaling molecules by H pylori CagA may predispose cells to accumulate multiple genetic and epigenetic changes that promote multistep gastric carcinogenesis. Intriguingly, the structural polymorphism of CagA accounts for the differences in pathophysiological activity of individual CagA proteins, raising the possibility of subclassification of H pylori strains into benign and malignant strains.

Antigens, Bacterial↗
PubMed2001

Circulating forms of parathyroid hormone detected with an immunofluorometric assay in patients with primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal failure.

In patients with uremia, intact parathyroid hormone (PTH) measurement appears to overestimate the biologically active hormone in circulation. The recent description of the accumulation in these patients of a non-intact PTH form measured by the standard immunometric assays, re-opened the question. In this study we submitted serum samples from 7 patients with primary hyperparathyroidism (PHP) and from 10 patients with hyperparathyroidism secondary to chronic renal failure (SHP) to preparative HPLC in order to discriminate the molecular forms measured by our currently used immunofluorometric assay for intact PTH. The elution profile obtained with the HPLC system showed two clearly defined peaks, the first one corresponding to a lower molecular weight form, and the second to the intact PTH (1-84) form. In patients with SHP the area under the curve for the first peak (mean 29.5%, range 20.6 to 40.4%) was significantly greater than that observed for patients with PHP (mean 15.6%, range 5.6 to 21.9%). This confirms previous studies showing accumulation of molecular forms of slightly lower molecular weight, presumably PTH (7-84), in patients with SHP and, to a lesser extent, in patients with PHP. The real necessity of assays that discriminate between these two molecular forms is debatable.

Chromatography, High Pressure Liquid↗
PubMed1997

Evidence for involvement of IgA1 hinge glycopeptide in the IgA1-IgA1 interaction in IgA nephropathy.

The study was performed to investigate the role of the IgA1 hinge region in the IgA1-IgA1 interaction, which was observed previously in IgA nephropathy. The competitive inhibition assays of the IgA1-IgA1 binding were performed using the following candidates for inhibitors: native IgA1 hinge glycopeptide (nHGP), IgA1, IgA2, and IgG. The IgA1-IgA1 binding was definitely inhibited by the nHGP and the IgA1 (maximum of percent inhibition: 66.1 and 60.5%, respectively). There was no obvious inhibition in the IgA2 and the IgG. The inhibition curves of the nHGP and the IgA1 were significantly different from that of the IgG (P < 0.01, respectively). Furthermore, to reveal the detailed binding sites in the interaction, the same inhibition assays were performed using the following substances composing the IgA1 hinge glycopeptide: galactose (Gal), N-acetyl-galactosamine (GalNAc), Gal beta 1-3GalNAc, sialic acid, tetrapeptide PTPS, and synthesized hinge proline-rich peptide PVPSTPPTPSPSTPPTPSPS (sHP). sHP, Gal beta 1-3GalNAc, Gal, and GalNAc inhibited the binding (69.3, 34.1, 14.9, 14.6%, respectively). No obvious inhibition was observed in sialic acid and tetrapeptide PTPS. The inhibition curve of sHP was significantly different from that of the PTPS (P < 0.05). Those of Gal beta 1-3GalNAc, Gal, and GalNAc were also significantly different from that of sialic acid (P < 0.05, respectively). These results suggested that the IgA1-IgA1 interaction could be mediated by the core structure including the peptide and the sugars, except for sialic acid in the hinge region, resulting in the formation of the circulating macromolecular IgA1 in IgA nephropathy.

Adult↗
PubMed1995

Summer-type hypersensitivity pneumonitis.

Summer-type hypersensitivity pneumonitis (SHP), the most prevalent type of HP in Japan, is caused by seasonal mold contamination in the home environment. The causative agent of the disease is Trichosporon cutaneum. The fungus grows in warm, moldy, decaying organic matter, and scatters in the air from the colonizing places. The inhaled fungi sensitize susceptible patients intratracheally and induce the disease. Glucuronoxylomannan of the fungus has a potent antigenicity that causes granulomatous alveolitis. Assay of anti-T. cutaneum antibody is very useful to establish the diagnosis of the disease because the antibody activity is virtually positive in all cases of the disease. Elimination of T. cutaneum from the colonizing places prevents recrudescence. SHP, a new form of HP, had been considered to be peculiar to Japan, but the first case of SHP outside Japan was identified in Korea last year. Soon it will be recognized in many countries of temperate and tropical clime.

Alveolitis, Extrinsic Allergic↗
PubMed2003

[Intraspecies diversity of Trichosporon asahii as the causative agent of opportunistic fungal infection and summer-type hypersensitivity pneumonitis].

Trichosporon asahii is the major causative agent of the opportunistic infection trichosporonosis, and also causes summer-type hypersensitivity pneumonitis (SHP). Random amplification of polymorphic DNA analysis was used to determine the intraspecies diversity of T. asahii isolates from clinical specimens,the houses of SHP patients, and environmental material. Clinical isolates formed a cluster, characterized by a 90% matching coefficient, but they did not cluster with strains isolated from SHP patients houses or environmental sources. The biochemical characteristics of T. asahii isolates from the three sources were compared, and a phenogram was constructed. One of the two clusters included most of the clinical isolates and strains isolated from the houses, and the other cluster included most of the environmental isolates. There was a remarkable difference in the abilities of the strains belonging to these clusters to utilize several compounds. These results suggest that the genetic diversity and biochemical characteristics of T. asahii are related to the source of the isolates. In addition, based on the IGS1 sequence, which is located between the 26S and 5S rRNA genes, we identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis. Of the five genotypes, three were isolates that originated in Japan, whereas two were American isolates. IGS sequence analysis shows great potential as a new epidemiological tool.

Alveolitis, Extrinsic Allergic↗
PubMed2001

Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions.

The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.

Cell Polarity↗
PubMed2002

Lyn and Syk kinases are sequentially engaged in phagocytosis mediated by Fc gamma R.

Recent data indicate that phagocytosis mediated by FcgammaRs is controlled by the Src and Syk families of protein tyrosine kinases. In this study, we demonstrate a sequential involvement of Lyn and Syk in the phagocytosis of IgG-coated particles. The particles isolated at the stage of their binding to FcgammaRs (4 degrees C) were accompanied by high amounts of Lyn, in addition to the signaling gamma-chain of FcgammaRs. Simultaneously, the particle binding induced rapid tyrosine phosphorylation of numerous proteins. During synchronized internalization of the particles induced by shifting the cell to 37 degrees C, Syk kinase and Src homology 2-containing tyrosine phosphatase-1 (SHP-1) were associated with the formed phagosomes. At this step, most of the proteins were dephosphorylated, although some underwent further tyrosine phosphorylation. Quantitative immunoelectron microscopy studies confirmed that Lyn accumulated under the plasma membrane beneath the bound particles. High amounts of the gamma-chain and tyrosine-phosphorylated proteins were also observed under the bound particles. When the particles were internalized, the gamma-chain was still detected in the region of the phagosomes, while amounts of Lyn were markedly reduced. In contrast, the vicinity of the phagosomes was heavily decorated with anti-Syk and anti-SHP-1 Abs. The local level of protein tyrosine phosphorylation was reduced. The data indicate that the accumulation of Lyn during the binding of IgG-coated particles to FcgammaRs correlated with strong tyrosine phosphorylation of numerous proteins, suggesting an initiating role for Lyn in protein phosphorylation at the onset of the phagocytosis. Syk kinase and SHP-1 phosphatase are mainly engaged at the stage of particle internalization.

Animals↗
PubMed2003

Curcumin suppresses Janus kinase-STAT inflammatory signaling through activation of Src homology 2 domain-containing tyrosine phosphatase 2 in brain microglia.

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that the inhibitory action of curcumin on Janus kinase (JAK)-STAT signaling can contribute to its anti-inflammatory activity in the brain. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the ganglioside-, LPS-, or IFN-gamma-stimulated induction of cyclooxygenase-2 and inducible NO synthase, important enzymes that mediate inflammatory processes. These anti-inflammatory effects appear to be due, at least in part, to the suppression of the JAK-STAT inflammatory signaling cascade. Curcumin markedly inhibited the phosphorylation of STAT1 and 3 as well as JAK1 and 2 in microglia activated with gangliosides, LPS, or IFN-gamma. Curcumin consistently suppressed not only NF binding to IFN-gamma-activated sequence/IFN-stimulated regulatory element, but also the expression of inflammation-associated genes, including ICAM-1 and monocyte chemoattractant protein 1, whose promoters contain STAT-binding elements. We further show that activation of Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-2, a negative regulator of JAK activity, is likely to be one of the mechanisms underlying the curcumin-mediated inhibition of JAK-STAT signaling. Treatment of microglial cells with curcumin led to an increase in phosphorylation and association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells.

Animals↗
PubMed2003

The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling.

The Src family kinase Lyn has been shown to play both stimulatory and inhibitory roles within several hemopoietic cell types. In this study, we investigated the role played by Lyn in neutrophil integrin signaling. Loss of Lyn resulted in a hyperresponsive phenotype on engagement of surface integrins at low valency. Lyn(-/-) neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when adherent to surfaces coated with either cellular counterreceptors or extracellular matrix proteins. In contrast, Lyn-deficient and wild-type cells expressed similar levels of surface integrins and responded equivalently to activating agents in suspension, indicating that the enhanced responses of lyn(-/-) cells was specific to the integrin signaling pathways. Lyn-deficient macrophages also displayed a hyperadhesive phenotype. Biochemical analysis of macrophages from lyn(-/-) mice revealed that Lyn plays an essential role in the adhesion-dependent phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of the inhibitory receptors SIRP1alpha and PIR-B, which in turn recruit the phosphatase SHP-1. These observations suggest that reduced mobilization of SHP-1 to the membrane in lyn(-/-) neutrophils results in a hyperadhesive and hyperactive phenotype. This hypothesis is further supported by the fact that neutrophils from me(v)/me(v) mice, which have significantly reduced SHP-1 activity, are also hyperresponsive following integrin engagement. This is the first direct evidence using primary leukocytes from lyn(-/-) mice that this kinase functions as a negative regulator in integrin signaling.

Animals↗
PubMed2005

Sodium stibogluconate interacts with IL-2 in anti-Renca tumor action via a T cell-dependent mechanism in connection with induction of tumor-infiltrating macrophages.

IL-2 therapy results in 10-20% response rates in advanced renal cell carcinoma (RCC) via activating immune cells, in which the protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) is a key negative regulator. Based on finding that sodium stibogluconate (SSG) inhibited SHP-1, the anti-RCC potential and action mechanism of SSG and SSG/IL-2 in combination were investigated in a murine renal cancer model (Renca). Despite its failure to inhibit Renca cell proliferation in cultures, SSG induced 61% growth inhibition of Renca tumors in BALB/c mice coincident with an increase (2-fold) in tumor-infiltrating macrophages (Mphi). A combination of SSG and IL-2 was more effective in inhibiting tumor growth (91%) and inducing tumor-infiltrating Mphi (4-fold), whereas IL-2 alone had little effect. Mphi increases were also detected in the spleens of mice treated with SSG (3-fold) or SSG/IL-2 in combination (6-fold), suggesting a systemic Mphi expansion similar to those in SHP-deficient mice. T cell involvement in the anti-Renca tumor action of the combination was suggested by the observations that the treatment induced spleen IFN-gamma T cells in BALB/c mice, but failed to inhibit Renca tumor growth in athymic nude mice and that SSG treatment of T cells in vitro increased production of IFN-gamma capable of activating tumoricidal Mphi. The SSG and SSG/IL-2 combination treatments were tolerated in the mice. These results together demonstrate an anti-Renca tumor activity of SSG that was enhanced in combination with IL-2 and functions via a T cell-dependent mechanism with increased IFN-gamma production and expansion/activation of Mphi. Our findings suggest that SSG might improve anti-RCC efficacy of IL-2 therapy by enhancing antitumor immunity.

Animals↗
PubMed2005

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses.

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.

Blotting, Northern↗
PubMed2005

Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

B-Lymphocytes↗