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PubMedDate not supplied

New technique for superior hypogastric plexus block.

BACKGROUND AND OBJECTIVES: : The classical technique for blocking the superior hypogastric plexus (SHP) described by Plancarte sometimes renders the desired needle placement difficult. This article describes an alternative approach for blocking the SHP. METHODS: Three patients with pelvic pain from endometriosis underwent an anterior approach to block the SHP with fluoroscopic guidance. The ages of the patients ranged from 21 to 34 years, pain duration ranged from 2 to 4 years, and pain score on a visual analog scale of 0-10 ranged from 7 to 8. RESULTS: All patients had significant pain relief immediately after the block. The pain scores postblock ranged from 0 to 4/10. The duration of pain relief varied from 1 to 14 days. The contrast material localized at the L5 vertebral body in the posteroanterior and lateral views. CONCLUSION: We present a new approach to block the SHP with fluoroscopic guidance in patients with chronic benign pelvic pain.

Adult↗
PubMed1997

Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand.

SHP-1, a protein tyrosine phosphatase containing two tandem SH2 domains, is autoinhibited at rest by its N-terminal SH2 (N-SH2) domain. Relief from autoinhibition and a subsequent 10-60-fold increase in V(max) have been observed upon N-SH2 domain engagement by a specific phosphotyrosyl ligand or upon deletion of the SH2 domains to yield the catalytic PTPase domain. In this study, we observed that glycerol and propane-1,2-diols, at concentrations of 4-6 M, accelerated the k(cat) of the full length enzyme by 47-fold and of the PTPase domain by 8-fold. Glycerol also increases the rate of proteolytic cleavage between the SH2 and catalytic PTPase domains. In stopped-flow studies using p-nitrophenyl phosphate (pNPP) as a substrate, a burst of p-nitrophenolate in the full length enzyme was not observed; however, a 50-70% stoichiometric burst was observed with the PTPase domain. Rapid quench studies using [32P]pNPP as a substrate showed a very low level of covalent [32P]phosphocysteinyl enzyme intermediate accumulation: 0.06% in the full length enzyme and 1% in the PTP domain. Stimulation by glycerol reduced the accumulating levels of phosphocysteinyl enzyme in both cases of full length SHP-1 and the PTPase domain; however, glycerol is not acting as a cosubstrate since no glycerophosphate product was detectable. It is likely that, for full length SHP-1, with pNPP as a model substrate, enzyme-substrate complex (ES) accumulates in its basal autoinhibited state, whereas enzyme-product complex (EP(i)) accumulates in its pY ligand-bound activated state. Glycerol probably relaxes the compact structure of SHP-1 and the PTP domain, thereby accelerating the catalytic rates in both cases by increasing forward reaction rates of ES and EP(i).

Amino Acid Sequence↗
PubMed2003

Tyr-c[D-Orn-Tyr(Bzl)-Pro-Gly]: a novel antiproliferative acting somatostatin receptor agonist with mu-opioid receptor-sensitizing properties.

(1) Here, we introduce a beta-casomorphin-5-derived cyclic pentapeptide, cCD-2 (Tyr-cyclo[d-Orn-Tyr(Bzl)-Pro-Gly]), which inhibits the cell growth of a variety of human cancer cell lines. (2) This opioid-derived peptide possesses only low affinity for mu-receptors, but enhances the agonist binding to mu-receptors in vitro and potentiates the analgesic effect of morphin in vivo. The molecular mechanism of mu-receptor sensitization by cCD-2 is not yet known. (3) The antiproliferative effect of cCD-2 is independent of mu-, delta-, and kappa-receptors. (4) Using SH-SY5Y cells as model, we can demonstrate that cCD-2 specifically binds to somatostatin receptors and stimulates the activity of protein tyrosine phosphatases, which are early downstream targets of SST receptors. (5) In SH-SY5Y cells, cCD-2 specifically increases the activity of the cytosolic PTP SHP-2, stimulates the activity of mitogen-activated protein kinase (MAPK), and elevates the expression of the cyclin-dependent kinase inhibitor p21 (WAF1/Cip1), suggesting the involvement of SSTR1 receptor subtype in cCD-2 action in this cell type. (6) In COS-7 cells, for comparison, we found a stimulation of SHP-2 as well as SHP-1 in response to cCD-2. The activation of SHP-1, which is attributed to the SSTR2 receptor and negatively regulates the EGF receptor, corresponds with the ability of cCD-2 to inhibit the EGF-induced MAPK activation in COS-7 cells. (7) Our results show that in SH-SY5Y cells cCD-2 inhibits cell growth via the SSTR1 receptor-signalling pathway but may, in other cells, also use other SSTR subtypes and their signalling mechanisms. (8) cCD-2 represents a novel type of opioid-derived antiproliferative SST receptor agonist, which possesses low mu-receptor affinity but may induce mu-receptor sensitization and is structurally different from the hitherto known SST receptor agonists.

Cell Division↗
PubMed2002

Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes.

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr(612) and Tyr(941) (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr(896) (a Grb2 binding site); and (iii) Tyr(1229) (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr(1158) and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (>or=20 min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.

3T3 Cells↗
PubMed2004

The nuclear bile acid receptor FXR is activated by PGC-1alpha in a ligand-dependent manner.

The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.

Animals↗
PubMed2001

A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33.

The siglecs (sialic acid-binding Ig-like lectins) are a distinct subset of the Ig superfamily with adhesion-molecule-like structure. We describe here a novel member of the siglec protein family that shares a similar structure including five Ig-like domains, a transmembrane domain, and a cytoplasmic tail containing two ITIM-signaling motifs. Siglec-10 was identified through database mining of an asthmatic eosinophil EST library. Using the Stanford G3 radiation hybrid panel we were able to localize the genomic sequence of siglec-10 within the cluster of genes on chromosome 19q13.3-4 that encode other siglec family members. We have demonstrated that siglec-10 is an immune system-restricted membrane-bound protein that is highly expressed in peripheral blood leukocytes as demonstrated by Northern, RT-PCR and flow cytometry. Binding assays determined that the extracellular domain of siglec-10 was capable of binding to peripheral blood leukocytes. The cytoplasmic tail of siglec-10 contains four tyrosines, two of which are embedded in ITIM-signaling motifs (Y597 and Y667) and are likely involved in intracellular signaling. The ability of tyrosine kinases to phosphorylate the cytoplasmic tyrosines was evaluated by kinase assay using wild-type siglec-10 cytoplasmic domain and Y-->F mutants. The majority of the phosphorylation could be attributed to Y597 andY667. Further experiments with cell extracts suggest that SHP-1 interacts with Y667 and SHP-2 interacts with Y667 in addition to another tyrosine. This is very similar to CD33, which also binds the phosphatases SHP-1 and SHP-2, therefore siglec-10, as CD33, may be characterized as an inhibitory receptor.

Amino Acid Motifs↗
PubMed2003

The effects of cure of Helicobacter pylori infection on the signal transduction of gastric epithelial cells.

BACKGROUND: The CagA protein of Helicobacter pylori is directly injected from the bacteria into cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the gastric epithelial cells. Translocated CagA forms a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2, which plays an important role in mitogenic signal transduction in the host cells. AIM: We examined the effect of eradication therapy on the signal transduction pathway of gastric epithelial cells induced by the CagA protein of H. pylori. METHODS: Gastric biopsy samples were obtained from 20 H. pylori-positive atrophic gastritis patients before, and 3 months after, H. pylori infection eradication therapy, and subjected to immunoblot analysis to detect tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2. RESULTS: Tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 were detected in the gastric mucosa from H. pylori-positive atrophic gastritis patients. All H. pylori strains from these patients were cagA-positive type I strains. After curing H. pylori infection, the tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 disappeared from the gastric mucosa. CONCLUSION: The cure of infection reduces the stimulated signal transduction of gastric epithelial cells by the translocated CagA protein of H. pylori, and may confer a beneficial effect on the reduction of cancer risk.

Bacterial Proteins↗
PubMed2002

Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose.

BACKGROUND: Both high glucose (HG) and angiotensin II (Ang II) causes glomerular mesangial cell (GMC) growth and increased synthesis of matrix proteins like collagen IV contributing to diabetic nephropathy. We have recently found that exposure of vascular smooth muscle cells to HG augments the Ang II activation of the growth promoting JAK/STAT pathway. We hypothesized that Ang II activation of the JAK/STAT pathway is altered by HG in GMC, and that this pathway might be linked to the Ang II-induced growth and overproduction of collagen IV in GMC in HG conditions. METHODS: GMC were cultured under normal glucose (NG; 5.5 mmol/L) and HG (25 mmol/L) for 48 hours and stimulated with Ang II (0.1 micromol/L) for various times. GMC lysate was then immunoprecipitated and/or immunoblotted with SHP-1, SHP-2 and phosphospecific JAK2 and STAT antibodies. The HG and Ang II induced growth and collagen IV synthesis studies were performed in GMC transfected with JAK2 antisense or JAK2 sense. GMC growth was monitored via [3H]-thymidine incorporation, and collagen IV synthesis via ELISA. RESULTS: We found that Ang II-induced JAK2, STAT1, STAT3, STAT5A/B and SHP-2 phosphorylations were enhanced by HG, whereas that of SHP-1 was reduced. Ang II-induced growth and collagen IV synthesis also were increased under HG conditions. Transfection of GMC with JAK2 antisense oligonucleotides blocked the Ang II-induced growth and collagen IV synthesis in both NG and HG conditions. CONCLUSION: These results provide evidence that activation of the JAK/STAT pathway by HG or/and Ang II may be of importance in the increased GMC cell growth and collagen IV synthesis that is seen in diabetic nephropathy.

Angiotensin II↗
PubMed2006

Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA.

BACKGROUND & AIMS: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. METHODS: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. RESULTS: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. CONCLUSIONS: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.

Antigens, Bacterial↗
PubMedDate not supplied

Coaxial imaging technique for superior hypogastric plexus block.

BACKGROUND AND OBJECTIVES: Previously described techniques for superior hypogastric plexus (SHP) block are technically difficult. A simple coaxial imaging technique for SHP block is described. METHODS AND RESULTS: Fluoroscopic guidance for coaxial approach to SHP block is described. A successful block is easily achieved. CONCLUSIONS: A new approach to SHP block is described, which is technically effective. Other approaches involve more difficult needle angulations or use of computed tomography guidance. The coaxial technique avoids these issues and is easy to perform.

Humans↗
PubMed2000

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils.

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.

Basophils↗
PubMed2003

The inhibitory potential of Fc receptor homolog 4 on memory B cells.

Fc receptor homolog 4 (FcRH4) is a B cell-specific member of the recently identified family of FcRHs whose intracellular domain contains three potential immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The signaling potential of this receptor, shown here to be preferentially expressed by memory B cells, was compared with the inhibitory receptor FcgammaRIIb in B cells expressing either WT FcgammaRIIb or chimeric proteins in which the intracellular domain of FcRH4 was fused to the transmembrane and extracellular domains of FcgammaRIIb. Coligation of the FcgammaRIIb/FcRH4 chimeric protein with the B cell receptor (BCR) led to tyrosine phosphorylation of the two membrane-distal tyrosines and profound inhibition of BCR-mediated calcium mobilization, whole cell tyrosine phosphorylation, and mitogen-activated protein (MAP)-kinase activation. Mutational analysis of the FcRH4 cytoplasmic region indicated that the two membrane-distal ITIMs are essential for this inhibitory potential. Phosphopeptides corresponding to these ITIMs could bind the Src homology 2 (SH2) domain-containing tyrosine phosphatases SHP-1 and SHP-2, which associated with the WT FcRH4 and with mutants having inhibitory capability. These findings indicate the potential for FcRH4 to abort B cell receptor signaling by recruiting SHP-1 and SHP-2 to its two membrane distal ITIMs.

Amino Acid Motifs↗
PubMed1997

Mapping of a cytoplasmic domain of the human growth hormone receptor that regulates rates of inactivation of Jak2 and Stat proteins.

It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.

Animals↗
PubMed1998

Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases.

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. Recently, it was observed that PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 was shown to associate with SHP-2, a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, we wished to understand better the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, we determined that mouse PECAM-1 could be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs, but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, we found that these PTKs were efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylated mouse PECAM-1 at one or more yet to be identified sites. In other studies, we demonstrated that phosphorylation of PECAM-1 by Src or Csk family kinases was sufficient to trigger its association with SHP-2. Moreover, it was able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicated that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances.

Amino Acid Sequence↗
PubMed1999

Integrins enhance platelet-derived growth factor (PDGF)-dependent responses by altering the signal relay enzymes that are recruited to the PDGF beta receptor.

Since the extracellular matrix (ECM) can promote platelet-derived growth factor (PDGF)-dependent responses, we hypothesized that the ECM mediates this effect by preventing the PDGF beta receptor (betaPDGFR) from associating with the negative regulator, RasGAP (the GTPase-activating protein of Ras). We found that binding of RasGAP to the wild-type betaPDGFR was decreased; the activation of Ras and Erk was enhanced, and [3H]thymidine uptake was better in cells cultured on fibronectin than in cells cultured on polylysine. To investigate the mechanism by which culturing cells on fibronectin diminished the recruitment of RasGAP to the betaPDGFR, we focused on SHP-2 since it dephosphorylates the betaPDGFR at the phosphotyrosine required for binding of RasGAP. Culturing cells on fibronectin increased the amount of SHP-2 that associated with the betaPDGFR. Furthermore, cells expressing receptor mutants that failed to associate with SHP-2 were insensitive to fibronectin. The ECM enhances PDGF-dependent responses by increasing the association of SHP-2 with the betaPDGFR, which in turn decreases the time that RasGAP interacts with the receptor. Thus, fibronectin changes PDGF-dependent signaling and biological responses by altering the signal relay enzymes that are recruited to the receptor.

Animals↗
PubMed2000

Activation of downstream signals by the long form of the leptin receptor.

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Crystal structures of an oxygen-binding cytochrome c from Rhodobacter sphaeroides.

The photosynthetic bacterium Rhodobacter sphaeroides produces a heme protein (SHP), which is an unusual c-type cytochrome capable of transiently binding oxygen during autooxidation. Similar proteins have not only been observed in other photosynthetic bacteria but also in the obligate methylotroph Methylophilus methylotrophus and the metal reducing bacterium Shewanella putrefaciens. A three-dimensional structure of SHP was derived using the multiple isomorphous replacement phasing method. Besides a model for the oxidized state (to 1.82 A resolution), models for the reduced state (2.1 A resolution), the oxidized molecule liganded with cyanide (1. 90 A resolution), and the reduced molecule liganded with nitric oxide (2.20 A resolution) could be derived. The SHP structure represents a new variation of the class I cytochrome c fold. The oxidized state reveals a novel sixth heme ligand, Asn(88), which moves away from the iron upon reduction or when small molecules bind. The distal side of the heme has a striking resemblance to other heme proteins that bind gaseous compounds. In SHP the liberated amide group of Asn(88) stabilizes solvent-shielded ligands through a hydrogen bond.

Bacterial Proteins↗
PubMed2000

Negative regulation of growth hormone receptor/JAK2 signaling by signal regulatory protein alpha.

Signal regulatory proteins (SIRPs) are receptor-like transmembrane proteins, the majority of which contain a cytoplasmic proline-rich region and four cytoplasmic tyrosines that, when phosphorylated, bind SH2 domain-containing protein tyrosine phosphatases (SHP). We demonstrated previously that growth hormone (GH) induces tyrosyl phosphorylation of SIRPalpha and association of SIRPalpha with SHP-2. The GH-activated tyrosine kinase JAK2 associates with and tyrosyl-phosphorylates SIRPalpha1. Here we show that JAK2-SIRPalpha1 association does not require phosphotyrosines in SIRPalpha1 or JAK2 or the proline-rich region of SIRPalpha1. However, when the C-terminal 30 amino acids of SIRPalpha1 containing the proline-rich region and tyrosine 495 are deleted, tyrosyl phosphorylation of SIRPalpha1 by JAK2 and association of SHP-2 with SIRPalpha1 are reduced. GH-dependent tyrosyl phosphorylation of JAK2 is reduced when wild-type SIRPalpha1 compared with SIRPalpha1 lacking the four cytoplasmic tyrosines (SIRP 4YF) is expressed in cells, suggesting that SIRPalpha1 negatively regulates GHR/JAK2 signaling. Consistent with reduced JAK2 activity, overexpression of wild-type SIRPalpha1 but not SIRP 4YF reduces GH-induced phosphorylation of ERKs 1 and 2, STAT3, and STAT5B. These results suggest that SIRPalpha1 is a negative regulator of GH signaling and that the ability of SIRPalpha1 mutants to negatively regulate GHR-JAK2 signaling correlates with their ability to bind SHP-2.

3T3 Cells↗