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PubMed2001

The effect of femoral prosthesis design on cement strain in cemented total hip arthroplasty.

Finite element studies show that the highest cement stresses are located at the most proximal and distal ends of the prosthesis. In vitro biomechanical and histologic analyses of autopsy-retrieved cemented femoral components show these areas to be associated with cement-prosthesis debonding. In this study, cement strains were measured in 2 geometrically different femoral stems in paired cadaver femora: A straight, collared, moderately tapered stem (Centralign) was compared with an anatomically curved, collarless, dramatically tapered stem (Scientific Hip Prosthesis [SHP]). Results showed that the maximum strain and the overall strain profile differed between the 2 stems. The Centralign had peak strains located at the most proximal gauge positions, whereas the peak strains of the SHP were located around the middle of the femoral stem. Minimization of cement strain, especially at the crucial proximal and distal areas of the stem, by altering component design may be able to reduce cement-prosthesis debonding and improve clinical results.

Arthroplasty, Replacement, Hip↗
PubMed1997

[The first manifestations of Schönlein-Henoch purpura in a 74-year-old female patient with hyperthyroidism].

HISTORY AND CLINICAL FINDINGS: A 74-year-old woman had for 3 weeks suffered from watery diarrhoea and diffuse abdominal pain. She felt restless, had a subfebrile temperature (37.8 degrees C), tachycardia and a blood pressure of 190/90 mmHg. Shortly after admission petechiae were found over the lower legs and she complained of joint pains. INVESTIGATIONS: Laboratory tests established hyperthyroidism. Skin biopsy showed leukocytoclastic vasculitis. Tests for antinuclear antibodies, antistreptolysin titre, rheumatoid factors, cryoglobulins and TSH-receptor antibodies were negative, immunoglobulin A (IgA) was raised. TREATMENT AND COURSE: As a drug-induced vasculitis was suspected treatment was started with methylprednisolone, 100 mg daily. Proteinuria (5.6 g daily) indicated renal biopsy, which revealed focal glomerulonephritis with deposits of IgA, fibrin/fibrinogen and complement factor 3. Gastroscopic biopsy, performed after an episode of gastrointestinal bleeding, demonstrated necrotizing vasculitis, confirming the diagnosis of Schönlein-Henoch-purpura (SHP). As the patient's condition rapidly worsened, cyclophosphamide was started additionally (2 mg/kg). She died on the 17th hospital day from acute cardiac failure. CONCLUSION: The lethal course of the disease in this elderly patient illustrates a previously not reported close temporal and clinical relationship between SHP and hyperthyroidism.

Abdominal Pain↗
PubMed1998

Partial splenectomy, transposition of the spleen to the abdominal wall or splenohepatoplasty in portal hypertensive rats. Effects on portal venous pressure and homeostasis--microscopical appearance of the transposed spleen.

Reduction of the splenic volume by partial resection and collateral development after transposition are of potential value in the elective treatment of esophageal varices, hypersplenism and ascites. A study was performed on young Wistar rats. A simple animal model of extrahepatic portal hypertension was used, narrowing the portal vein to an outer diameter of one millimeter (PVS). One day, three weeks and seven weeks after this operation the portal venous pressure was elevated as compared with the sham-operated controls. The portal hypertension was statistically significant at week three (1.31 +/- 0.04 vs. 0.72 +/- 0.18 kPa, p = 0.01). Portocaval pressure gradient after partial resection of the spleen (SR) and intramuscular transposition (IMTrans) was compared with the pressure gradient after graded portal vein stenosis. Three weeks after intramuscular transposition portocaval pressure gradient was reduced (1.46 +/- 0.38 vs. 1.74 +/- 0.13 kPa, n.s.). This data supports the hypothesis that the portal venous high-pressure compartment and the systemic venous low-pressure compartment are maintained after development of natural shunts to the systemic circulation. In the following experiment different types of splenic transposition were tested and compared to each other and a normal control group or to rats with protal vein stenosis (PVS), respectively. After PVS, the animals were reoperated, an SR was performed and the wound surface of the spleen was transposed into the left abdominal wall subcutaneously (SCTrans) or intramuscularly (IMTrans) or to the left liver lobe (splenohepatoplasty, SHP), respectively. After three weeks the animals underwent measurements of organ weights, collections of blood samples and the spleen was investigated histologically. Blood cell counts were nearly normal but total serum protein, albumin and the colloid osmotic pressure were slightly diminished or significantly reduced (COP in the groups PVS + SR + IMTrans or SCTrans, p < 0.05) compared to the controls. Differences to the group with portal vein stenosis (PVS) were not significant. Serum protein electrophoresis after splenic transposition revealed an unobtrusive distribution pattern. Animals after SHP had the best increase in weight and high protein levels, but splenohepatic collateral development seemed sparse. Differences of plasma ammonium levels were statistically not significant, but some animals had elevated levels after transposition. Morphometry of routine-stained spleen specimens showed an intact immunoarchitecture of the transposed spleens.

Abdominal Muscles↗
PubMed2000

IL-4/IL-13 signaling beyond JAK/STAT.

In the past several years, extensive studies on the mechanisms underlying IL-4 and IL-13 signaling have enabled us to gain insight into how these cytokines regulate immune responses. Because both IL-4 and IL-13 use the IL-4Ralpha as a receptor component, these cytokines activate many common signaling pathways. Both of these cytokines use Janus kinases (JAKs) to initiate signaling and activate signal transducer and activator of transcription-6 (STAT6), which is a transcription factor required for many of their biologic functions. In addition to JAK/STAT, these cytokines also activate a variety of other signaling molecules that are important in regulating IL-4-induced proliferation and protection from apoptosis. Suppressor of cytokine signaling-1 (SOCS-1) is a molecule that can inhibit the activation of IL-4 signaling through the inhibition of JAKs. The Fes tyrosine kinase is activated by IL-4 and appears to be important in regulating IL-4-induced proliferation through the phosphorylation of insulin receptor substrate (IRS) molecules. IRS molecules are essential for IL-4-induced proliferation through their ability to recruit phosphoinositol-3 kinase to the activated IL-4 receptor kinase. In addition, IL-4 can activate a number of phosphatases including SH2-containing inositol phosphatase (SHIP), SHP-1, and SHP-2. Finally, B-cell lymphoma gene-6 (BCL-6) appears to regulate a subset of IL-4-induced genes. Thus the biologic responses induced by IL-4/IL-13 require a complex interaction of signaling pathways and regulators.

Carrier Proteins↗
PubMed2004

Ly49Q, a member of the Ly49 family that is selectively expressed on myeloid lineage cells and involved in regulation of cytoskeletal architecture.

Here, we identified and characterized a Ly49 family member, designated as Ly49Q. The Ly49q gene encodes a 273-aa protein with an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N terminus of its cytoplasmic domain. We show that the ITIM of Ly49Q can recruit SHP-2 and SHP-1 in a tyrosine phosphorylation-dependent manner. In contrast to other known members of the Ly49 family, Ly49Q was found not to be expressed on NK1.1(+) cells, but instead was detectable on virtually all Gr-1(+) cells, such as myeloid precursors in bone marrow. Monocytes/macrophages also expressed low levels of Ly49Q, and the expression was enhanced by the treatment of cells with IFN-gamma. Treatment of activated macrophages with anti-Ly49Q mAb induced rapid formation of polarized actin structures, showing filopodia-like structure on one side and lamellipodial-like structure on the other side. A panel of proteins became tyrosine-phosphorylated in myeloid cells when treated with the mAb. Induction of the phosphorylation depends on the ITIM of Ly49Q. Thus, Ly49Q has unique features different from other known Ly49 family members and appears to be involved in regulation of cytoskeletal architecture of macrophages through ITIM-mediated signaling.

Animals↗
PubMed2001

PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.

PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (Fc gamma RIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca(2+) mobilization, and tyrosine phosphorylation of effector molecules, including Ig beta, Syk, phospholipase C-gamma 2 (PLC gamma 2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling.

Animals↗
PubMed1998

LCK-phosphorylated human killer cell-inhibitory receptors recruit and activate phosphatidylinositol 3-kinase.

HLA-specific killer cell inhibitory receptors (KIR) are thought to impede natural killer (NK) and T cell activation programs through recruitment of the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, to their cytoplasmic tails (CYT). To identify other SH2 domain-containing proteins that bind KIR CYT, we used the recently described yeast two-bait interaction trap and a modified version of this system, both of which permit tyrosine phosphorylation of bait proteins. Using these systems, we show that KIR CYT, once phosphorylated by the src-family tyrosine kinase LCK, additionally bind the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. Furthermore, we show that in an NK cell line, NK3.3, cross-linking of KIR results in recruitment of p85alpha to KIR and activation of PI 3-kinase lipid kinase activity. One consequence of KIR coupling to PI 3-kinase is downstream activation of the antiapoptotic protein kinase AKT. Therefore, in addition to providing negative signals, KIR may also contribute positive signals for NK and T cell growth and/or survival.

Cell Line↗
PubMed1999

The tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein kinase Akt/PKB in B lymphocytes.

The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1997

Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.

Animals↗
PubMed1997

Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney.

The intraperitoneal injection of a vanadate/H2O2 mixture (peroxovanadate) into mice resulted within minutes in the appearance of numerous tyrosine-phosphorylated proteins in the liver and kidney. These effects are presumably due to the inhibition of phosphotyrosine phosphatase activity. Three of the tyrosine-phosphorylated proteins have been identified as the receptors for epidermal growth factor, insulin, and hepatocyte growth factor. The injection of peroxovanadate also enhanced the tyrosine phosphorylation of many of the proteins known to function downstream of these receptors, including SHC, signal transducer and activator of transcription (Stat) 1alpha,beta, Stat 3, Stat 5, phospholipase C-gamma, insulin receptor substrate 1, GTPase-activating protein, beta-catenin, gamma-catenin, p120cas, SHP-1, and SHP-2. The administration of peroxovanadate also induced nuclear translocation of a number of tyrosine-phosphorylated Stat proteins. In addition, the global effects on tyrosine phosphorylation permitted the detection of a number of novel intracellular protein interactions, including an association of Tyk2 with beta-catenin. The in situ administration of peroxovanadate may prove useful in the search for novel tyrosine-phosphorylated proteins and the identification of new interactions between previously identified tyrosine-phosphorylated substrates.

Animals↗
PubMed1997

The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells.

Vascular endothelial growth factor A (here referred to as VEGF) is an endothelium-specific growth factor that binds to two distinct receptor tyrosine kinases, designated Flt-1 and KDR/Flk-1. VEGF stimulates autophosphorylation of both receptors, but little is known about their signal transduction properties. In this study, we used porcine aortic endothelial (PAE) cells overexpressing KDR (PAE/KDR) to evaluate the interaction of KDR with intracellular proteins and compared them with Flt-1-expressing PAE cells (PAE/Flt-1). VEGF-induced stimulation of KDR results in the association and phosphorylation of the 46-, 52-, and 66-kDa isoforms of Shc and the induction of Shc-Grb2 complex formation. In a similar fashion, KDR associates with Grb2 and Nck in a ligand-dependent fashion, suggesting Shc, Grb2, and Nck as potential candidates involved in the regulation of endothelial function. Another strong candidate is mitogen-activated protein (MAP) kinase, which is strongly activated in response to VEGF stimulation as demonstrated by phosphorylation of the specific substrate myelin basic protein. Inhibition of MAP kinase activation by PD98059, a specific MAP kinase kinase inhibitor, results in inhibition of VEGF-induced proliferation of PAE/KDR cells. In contrast, VEGF-induced stimulation of Flt-1 does not activate MAP kinase in PAE/Flt-1 cells. In this study we provide the first two examples of molecules potentially capable of functionally counteracting the endothelial response to VEGF, namely SHP-1 and SHP-2. These two SH2 protein-tyrosine phosphatases physically associate with KDR secondary to VEGF stimulation, raising the interesting possibility that both molecules participate in the generation and/or modulation of VEGF-induced signals. Taken together, our results substantially broaden the spectrum of KDR-associating molecules, indicating that endothelial function and angiogenesis are regulated by a diverse network of signal transduction cascades.

Animals↗
PubMed1998

Platelet-derived growth factor stimulates protein kinase D through the activation of phospholipase Cgamma and protein kinase C.

Platelet-derived growth factor (PDGF) stimulates protein kinase D (PKD) in a time- and dose-dependent manner. We have used a series of PDGF receptor mutants that display a selective impairment of the binding of SH2-containing proteins (GTPase-activating protein, SHP-2, phospholipase Cgamma (PLCgamma), or phosphatidylinositol 3'-kinase (PI3K)) to show that Tyr-1021, the PLCgamma-binding site, is essential for PKD stimulation by PDGF in A431 cells. We next investigated whether any one of these four binding sites could mediate PKD activation in the absence of the other three sites. F5, a receptor mutant that lacks all four binding sites for GTPase-activating protein, PLCgamma, PI3K, and SHP-2, fails to activate PKD. A panel of single add-back mutants was used to investigate if any one of these four sites could restore signaling to PKD. Of the four sites, only the PLCgamma+ single add-back receptor restored PDGF-mediated activation of PKD, and only this add-back receptor produced diacylglycerol (DAG) in a PDGF-dependent manner. 1,2-Dioctanoyl-sn-glycerol, a membrane-permeant DAG analog, was found to be sufficient for activation of PKD. Taken together, these data indicate that PLCgamma activation is not only necessary, but also sufficient to mediate PDGF-induced PKD activation. Although the presence of a pleckstrin homology domain makes PKD a potential PI3K target, PKD was not stimulated by selective PI3K activation, and wortmannin, an inhibitor of PI3K, did not inhibit PDGF signaling to PKD. The activation of PKD by DAG or by the wild-type and PLCgamma+ add-back PDGF receptors was inhibited by GF109203X, suggesting a role for protein kinase C in the stimulation of PKD by PDGF. PDGF induced a time-dependent phosphorylation of PKD that closely correlated with activation. The PDGF-induced activation and phosphorylation of PKD were reversed by in vitro incubation of PKD with protein phosphatase 1 or 2A, indicating that PDGF signaling to PKD involves the Ser/Thr phosphorylation of PKD. Taken together, these results conclusively show that PDGF activates PKD through a pathway that involves activation of PLCgamma and, subsequently, protein kinase C.

3T3 Cells↗
PubMed1998

Colony-stimulating factor-1 stimulates the formation of multimeric cytosolic complexes of signaling proteins and cytoskeletal components in macrophages.

Stimulation of macrophages with colony-stimulating factor-1 (CSF-1) results in the protein tyrosine phosphorylation of the CSF-1 receptor (CSF-1R) and many other, primarily cytosolic, proteins. Stimulation by CSF-1 at 4 degreesC was used to facilitate the purification and identification of the proteins of the cytosolic anti-phosphotyrosine (PY)-reactive fraction (alphaPY-RF) involved in downstream signaling pathways. Confocal microscopy revealed that the PY proteins are in close proximity to the CSF-1R at the plasma membrane. The alphaPY-RF contained pre-existing complexes of PY proteins and non-PY proteins which generally increased in size and PY protein content following CSF-1 stimulation. PY proteins identified by microsequencing and Western blotting include Cbl, STAT3, STAT5a, STAT5b, SHP-1, Shc, and two novel proteins pp57 and pp37. Other proteins included cytoskeletal/contractile proteins (paxillin, vimentin, elongation factor-1alpha, F-actin, tropomyosin, and myosin regulatory light chain), Ras family signaling proteins (p85 (phosphoinositide 3-kinase), Vav, Ras-GTPase-activating protein SH3 domain-binding protein, and Grb2), DnaJ-like protein, and glyceraldehyde-3-phosphate dehydrogenase. CSF-1 induced the de novo recruitment of Cbl, STAT3, STAT5a, STAT5b, p85, SHP-1, Shc, vimentin, and Grb2 to complexes and caused pre-existing complexes involving Vav, elongation factor-1alpha, and F-actin to increase in size. These studies indicate that CSF-1-induced protein tyrosine phosphorylation is associated with the reorganization of complexes of cytoskeletal, signaling, and other proteins that mediate CSF-1-regulated motility and growth.

Actins↗
PubMed1998

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules.

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.

3T3 Cells↗
PubMed1999

Disruption of gap junctional communication by the platelet-derived growth factor is mediated via multiple signaling pathways.

The platelet-derived growth factor (PDGF) mediates its cellular functions via activation of its receptor tyrosine kinase followed by the recruitment and activation of several signaling molecules. These signaling molecules then initiate specific signaling cascades, finally resulting in distinct physiological effects. To delineate the PDGF signaling pathway responsible for the disruption of gap junctional communication (GJC), wild-type PDGF receptor beta (PDGFRbeta) and a series of PDGFRbeta mutants were expressed in T51B rat liver epithelial cells. In cells expressing wild-type PDGFRbeta, PDGF induced disruption of GJC and phosphorylation of a gap junctional protein, connexin-43 (Cx43), which required activation of mitogen-activated protein kinase, although involvement of additional factors was also evident. In the F5 mutant lacking binding sites for phosphatidylinositol 3-kinase, GTPase-activating protein, SHP-2, and phospholipase Cgamma1 (PLCgamma1), PDGF induced mitogen-activated protein kinase, but failed to affect GJC or Cx43, indicating involvement of additional signals presumably initiated by one or more of the mutated binding sites. Examination of the single-site mutants revealed that PDGF effects were not mediated via a single signaling component. This was confirmed by the "add-back" mutants, which showed that restoration of either SHP-2 or PLCgamma1 binding was sufficient to propagate the GJC inhibitory actions of PDGF. Further analysis showed that activation of PLCgamma1 is involved in Cx43 phosphorylation, which surprisingly failed to correlate with GJC blockade. The results of our study demonstrate that PDGF-induced disruption of GJC can be mediated by multiple signaling pathways and requires participation of multiple components.

Animals↗
PubMed1999

Signal regulatory proteins negatively regulate immunoreceptor-dependent cell activation.

Signal regulatory proteins of the alpha subtype (SIRPalpha) are ubiquitous molecules of the immunoglobulin superfamily that negatively regulate protein tyrosine kinase receptor-dependent cell proliferation. Their intracytoplasmic domain contains four motifs that resemble immunoreceptor tyrosine-based inhibition motifs (ITIMs) and that, when tyrosyl-phosphorylated, recruit cytoplasmic SH2 domain-bearing protein tyrosine phosphatases (SHPs). ITIMs are borne by molecules that negatively regulate cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). Because SIRPalpha are coexpressed with ITAM-bearing receptors in hematopoietic cells, we investigated whether SIRPalpha could negatively regulate ITAM-dependent cell activation. We found SIRPalpha transcripts in human mast cells, and we show that a chimeric molecule having the transmembrane and intracytoplasmic domains of SIRPalpha could inhibit IgE-induced mediator secretion and cytokine synthesis by mast cells. Inhibition required that the SIRPalpha chimera was coaggregated with ITAM-bearing high affinity IgE receptors (FcepsilonRI). It was correlated with the tyrosyl phosphorylation of the SIRPalpha chimera and the recruitment of SHP-1 and SHP-2. The phosphorylation of FcepsilonRI ITAMs was decreased; the mobilization of intracellular Ca(2+) and the influx of extracellular Ca(2+) were reduced, and the activation of the mitogen-activated protein kinases Erk1 and Erk2 was abolished. SIRPalpha can therefore negatively regulate not only receptor tyrosine kinase-dependent cell proliferation but also ITAM-dependent cell activation.

Antigens, Differentiation↗
PubMed2000

Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein.

Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular interactions that influence the balance of these processes, especially the phosphorylation state of postinsulin receptor substrates, such as insulin receptor substrate-1 (IRS-1). The specific activity of four candidate protein-tyrosine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-containing PTPase-2 (SHP-2), leukocyte common antigen-related (LAR), and leukocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro. PTP1B exhibited the highest specific activity (percentage dephosphorylated per microg per min), and the enzyme activities varied over a range of 5.5 x 10(3). When evaluated as a ratio of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B remained significantly more active by 3.1-293-fold, respectively. Overlay blots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showed that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit of phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 dephosphorylation. Further studies revealed that the adaptor protein GRB2 strongly promoted the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immunoprecipitation from an equimolar solution by 13.5 +/- 3.3-fold (n = 7; p < 0.01). Inclusion of GRB2 in a reaction mixture of IRS-1 and active PTP1B also increased the overall rate of IRS-1 tyrosine dephosphorylation by 2.7-3.9-fold (p < 0.01). These results provide new insight into novel molecular interactions involving PTP1B and GRB2 that may influence the steady-state capacity of IRS-1 to function as a phosphotyrosine scaffold and possibly affect the balance of postreceptor insulin signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

SOCS3 targets Siglec 7 for proteasomal degradation and blocks Siglec 7-mediated responses.

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Animals↗